Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study.
Resnick Murray B,Konkin Tamako,Routhier Justin,Sabo Edmond,Pricolo Victor E
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Tight junction associated proteins are key molecular components governing cellular adhesion, polarity and glandular differentiation. Tight junction proteins also play critical roles in cellular proliferation and neoplastic pathways via their functions as couplers of the extracellular milieu to intracellular signaling pathways and the cytoskeleton. Neoplastic cells frequently exhibit structural and functional deficiencies in the tight junction. The purpose of this study was to determine the pattern of expression and prognostic value of four tight junction associated proteins, claudin-1, claudin-4, occludin and ZO-1 in a cohort of TNM stage II colon cancer using tissue microarray technology. In this study, we retrospectively analyzed, resected and otherwise untreated paraffin embedded specimens from 129 consecutive patients with TNM stage II colonic carcinomas for claudin-1, claudin-4, occludin and ZO-1 protein expression by immunohistochemistry. Seventy-five, 58, 56 and 44% of the tumors exhibited normal to elevated expression levels (+2 and +3 immunopositivity) of claudin-1, claudin-4, occludin and ZO-1 respectively. Low expression levels of claudin-1 and ZO-1 were directly associated with higher tumor grade (P=0.05 and 0.03 respectively). Multivariate analysis indicated that lymphovascular invasion (P=0.01) and low levels of claudin-1 (P=0.0001) expression were independent predictors of recurrence and that reduced claudin-1 expression (P=0.0001) was associated with poor survival. This study is the first to comprehensively examine the expression of several tight junction associated proteins in colonic neoplasms and to correlate their expression with disease progression. Loss of claudin-1 expression proved to be a strong predictor of disease recurrence and poor patient survival in stage II colon cancer.
Prognostic importance of Claudin-1 and Claudin-4 expression in colon carcinomas.
Ersoz Safak,Mungan Sevdegul,Cobanoglu Umit,Turgutalp Havvanur,Ozoran Yavuz
Pathology, research and practice
In this study, we analyzed Claudin-1 and Claudin-4 expressions in colon carcinomas. We investigated the relationship between the expression of these tight junction proteins and clinicopathologic parameters. Claudin-1 and 4 expressions were determined by immunohistochemical methods, and the rate of cells expressing these tight junction proteins were calculated with stereologic methods. Fifty-nine colon cancer cases were enrolled in the study group. Claudin-1 and 4 expressions were found to be significantly lower in cases with lymph node metastasis. Mean staining rates of Claudin-1 and 4 in lymph node (+) cases were 36.1±20.1 and 58±28.9, while in lymph node (-) cases, these were 63.8±25.9 and 72.3±25.6, respectively (p=0.0005 for Claudin-1, p=0.049 for Claudin-4). The mean staining rate for Claudin-1 in adenomatous polyps was significantly higher than incarcinomas (77.13±23.4 and 50.6±26.93, respectively) (p=0.003), while it was quite similar for Claudin-4 (65.4±26.9 and 65.3±27.9, respectively). In this study, we demonstrated Claudin-1 and 4 expressions in colon cancer cases. Claudin-1 expression seems to be more prominent in adenomatous polyps as compared with cancer cases. Expression of Claudin-1 decreases significantly in the presence of lymph node metastasis and diminishes in mucinous carcinoma cases, indicating a negative correlation between Claudin-1 expression and neoplastic progression.
Decreased expression of claudin-1 in rectal cancer: a factor for recurrence and poor prognosis.
Yoshida Takefumi,Kinugasa Tetsushi,Akagi Yoshito,Kawahara Akihiko,Romeo Kansakar,Shiratsuchi Ichitarou,Ryu Yasuhiko,Gotanda Yukito,Shirouzu Kazuo
AIM:To investigate the potential involvement of claudin-1 (CL-1) in the tumorigenesis of rectal cancer by analyzing the correlation between CL-1 expression, clinicopathological factors and prognosis. PATIENTS AND METHODS:Rectal cancer tissue specimens from 306 patients that had undergone surgical treatment were evaluated using immunohistochemical analysis for expression of CL-1 and correlated with clinicopathological factors. RESULTS:A reduced expression of CL-1 (less than 30% of tumor cells strongly, positively stained) correlated significantly with poor prognosis in stage II and III rectal cancer. Moreover, the expression levels of CL-1 correlated significantly with tumor differentiation and perineural invasion (p=0.037 and 0.009, respectively). However, no significant differences were detected between the expression levels of CL-1 and other clinicopathological factors. CONCLUSION:Loss of claudin-1 expression is a strong predictor of disease recurrence and poor patient survival in stage II and III rectal cancer.
Predictive value of immunohistochemical expression of claudin-1 in colonic carcinoma.
Abdelzaher Eman,Rizk Azza Mohamed,Bessa Samer Saad,Omer Khalafalla Mustafa
Journal of the Egyptian National Cancer Institute
OBJECTIVE:Colonic carcinoma is one of the most common cancers worldwide. Recently, the possible involvement of claudin-1, one of the major tight junction proteins, in the process of tumorigenesis has been suggested. Also, claudin-1 has emerged as a potential prognostic factor in different types of tumors. The aim of this study was to detect caludin-1 expression in colonic carcinoma and to correlate its expression with clinicopathological variables in an attempt to delineate its role as a potential new prognostic marker. MATERIAL AND METHODS:Immunohistochemical expression of claudin-1 was assessed in 50 Egyptian patients with colonic adenocarcinoma. The predictive performance of claudin-1 expression was statistically evaluated. RESULTS:Decreased claudin-1 expression was found in 62% of colonic adenocarcinoma cases while similar expression was found in 38% of the cases. Statistical analysis showed a statistically significant inverse correlation between claudin-1 expression and tumor grade, depth of invasion, lymph node involvement, and tumor stage. Regression analysis showed that claudin-1 decreased expression significantly predicts that the tumor is of a high grade, high stage, and is associated with lymph node involvement. ROC curve analysis showed that claudin-1 had a sensitivity of 88.24% and a specificity of 81.25% for the prediction of tumor stage and a sensitivity of 73.33% and a specificity of 82.86% for the prediction of lymph node involvement. CONCLUSIONS:Claudin-1 decreased expression in colonic carcinoma contributes to tumor dedifferentiation, invasion and metastasis. Claudin-1 expression could be used as a predictor of colonic carcinoma stage and lymph node status with a high sensitivity and specificity.
DNA demethylation of claudin-4 suppresses migration and invasion in laryngeal squamous carcinoma cells.
Liu Yafang,Chang Kai,Fu Kexin,Dong Xinjie,Chen Xiaoshuai,Liu Jixuan,Cui Ni,Ni Jinsong
Claudin-4 (CLDN4) is a member of the claudin transmembrane protein family, which consists of integral membrane proteins that are components of the epithelial cell tight junctions; these tight junctions regulate movement of solutes and ions through the paracellular space. CLDN4 is also a differentiation marker and is believed to indicate an epithelial phenotype. However, the role of CLDN4 in laryngeal squamous carcinoma is still unclear. Here, we showed that CLDN4 expression was down-regulated in laryngeal squamous carcinoma tissues and negatively correlated with methyl-CpG-binding protein 2. In addition, CLDN4 was hypermethylated in HEp-2 cells. DNA demethylation of CLDN4 by 5-aza-2'-deoxycytidine suppressed migration and invasion of HEp-2 cells, whereas CLDN4 silencing restored the migration and invasion of HEp-2 cells. Therefore, CLDN4 plays a key role in laryngeal squamous carcinoma progression.
Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer.
Dhawan Punita,Singh Amar B,Deane Natasha G,No Yiran,Shiou Sheng-Ru,Schmidt Carl,Neff John,Washington M Kay,Beauchamp R Daniel
The Journal of clinical investigation
Disruption of the cell-cell junction with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis. The role of adherent junction proteins has been studied extensively in cancer, but the roles of tight junction (TJ) proteins are less well understood. Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs. Recent studies show changes in expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established. Here, we report an increased expression of claudin-1 in human primary colon carcinoma and metastasis and in cell lines derived from primary and metastatic tumors. We also report frequent nuclear localization of claudin-1 in these samples. Genetic manipulations of claudin-1 expression in colon cancer cell lines induced changes in cellular phenotype, with structural and functional changes in markers of epithelial-mesenchymal transition. Furthermore, we demonstrate that changes in claudin-1 expression have significant effects on growth of xenografted tumors and metastasis in athymic mice. We further provide data suggesting that the regulation of E-cadherin expression and beta-catenin/Tcf signaling is a possible mechanism underlying claudin-1-dependent changes.
Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling.
Pope Jillian L,Bhat Ajaz A,Sharma Ashok,Ahmad Rizwan,Krishnan Moorthy,Washington Mary K,Beauchamp Robert D,Singh Amar B,Dhawan Punita
OBJECTIVE:Claudin-1 expression is increased and dysregulated in colorectal cancer and causally associates with the dedifferentiation of colonic epithelial cells, cancer progression and metastasis. Here, we have sought to determine the role claudin-1 plays in the regulation of intestinal epithelial homeostasis. DESIGN:We have used a novel villin-claudin-1 transgenic (Cl-1Tg) mouse as model (with intestinal claudin-1 overexpression). The effect of claudin-1 expression upon colonic epithelial differentiation, lineage commitment and Notch-signalling was determined using immunohistochemical, immunoblot and real-time PCR analysis. The frequently used mouse model of dextran sodium sulfate (DSS)-colitis was used to model inflammation, injury and repair. RESULTS:In Cl-1Tg mice, normal colonocyte differentiation programme was disrupted and goblet cell number and mucin-2 (muc-2) expressions were significantly downregulated while Notch- and ERK1/2-signalling were upregulated, compared with the wild type-littermates. Cl-1Tg mice were also susceptible to colonic inflammation and demonstrated impaired recovery and hyperproliferation following the DSS-colitis. Our data further show that claudin-1 regulates Notch-signalling through the regulation of matrix metalloproteinase-9 (MMP-9) and p-ERK signalling to regulate proliferation and differentiation. CONCLUSIONS:Claudin-1 helps regulate intestinal epithelial homeostasis through the regulation of Notch-signalling. An upregulated claudin-1 expression induces MMP-9 and p-ERK signalling to activate Notch-signalling, which in turn inhibits the goblet cell differentiation. Decreased goblet cell number decreases muc-2 expression and thus enhances susceptibility to mucosal inflammation. Claudin-1 expression also induces colonic epithelial proliferation in a Notch-dependent manner. Our findings may help understand the role of claudin-1 in the regulation of inflammatory bowel diseases and CRC.
Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy.
Pahle Jessica,Menzel Lutz,Niesler Nicole,Kobelt Dennis,Aumann Jutta,Rivera Maria,Walther Wolfgang
BACKGROUND:Bacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector. METHODS:In this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice. RESULTS:Claudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression. CONCLUSIONS:This novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo.
Claudin-1 role in colon cancer: An update and a review.
Histology and histopathology
Tight junction proteins are essential for sealing the cellular sheets and controlling para-cellular ion flux. Our understanding of the role that tight junction proteins, particularly claudins, play in cellular functions and pathologic conditions is continuously expanding. Particularly, the role of claudin-1 in oncogenesis in multiple locations in the human body is coming to light. This review will shed light on the role of claudin-1 in colon cancer. It will address the mechanisms through which claudin-1 becomes dysregulated in colon cancer. This will provide a platform to address results of claudin-1 expression in the third most common malignant tumour worldwide. Furthermore, it will provide updates about possible use of this biomarker in the surveillance of difficult colon maladies, such as inflammatory bowel disease. The use of claudin-1 as a biomarker of diagnostic and prognostic values will provide Medicine with much needed ammunition in the fight against cancer and will bring about, with added refinements, a new chapter in the era of personalized medicine to tackle this disease and match its destructive course with equally powerful and specifically targeted therapies.
Disturbed expression of E-cadherin, beta-catenin and tight junction proteins in colon carcinoma is unrelated to growth pattern and genetic polymorphisms.
Hahn-Strömberg Victoria,Edvardsson Henrik,Bodin Lennart,Franzén Lennart
APMIS : acta pathologica, microbiologica, et immunologica Scandinavica
Adhesion proteins are responsible for the structural integrity of epithelial tissue and in tumors this integrity is often lost, resulting in a disorganization of the tissue. In the present study the complexity of the invasive front of colon carcinomas was correlated with cell adhesion protein expression and with polymorphisms in their genes. A complexity index was constructed from 32 colon carcinomas using computer-assisted morphometry estimating fractal dimension and tumor cell clusters followed by tree analysis. Immunohistochemical staining of beta-catenin, E-cadherin, occludin and claudin 2 was used for assessment of protein expression. Genetic screening of tissue from the tumor invasion front with laser microdissection was performed using SSCP and DNA sequencing. Adhesion protein distribution was significantly disturbed in most carcinomas. A single mutation in the gene of beta-catenin was found but there was no correlation between protein expression and genetic polymorphism. Nor was there any correlation between the complexity of the invasive border and protein distribution or genetic alterations. The results indicate that the complexity of colon carcinoma invasion is not dependent on genetic derangements in the genes of adhesion proteins or the protein distribution. Rather, aberrations in the function of other proteins related to the adhesive proteins could be responsible.
Different expression of occludin and ZO-1 in primary and metastatic liver tumors.
Orbán Erika,Szabó Erzsébet,Lotz Gábor,Kupcsulik Péter,Páska Csilla,Schaff Zsuzsa,Kiss András
Pathology oncology research : POR
Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.
Anti-Claudin-1 Conjugated to a Near-Infrared Fluorophore Targets Colon Cancer in PDOX Mouse Models.
Hollandsworth Hannah M,Lwin Thinzar M,Amirfakhri Siamak,Filemoni Filemoni,Batra Surinder K,Hoffman Robert M,Dhawan Punita,Bouvet Michael
The Journal of surgical research
INTRODUCTION:Claudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery. METHODS:We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity. RESULTS:Western blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 μg of Claudin-1-IRDye800CW. CONCLUSIONS:Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.
Claudins regulate the intestinal barrier in response to immune mediators.
Kinugasa T,Sakaguchi T,Gu X,Reinecker H C
BACKGROUND & AIMS:To determine the functional role of immune mediators in the formation of the intestinal barrier, we have examined the regulation of claudin expression by interleukin (IL)-17 in human intestinal epithelial cells. METHODS:Expression of claudins, extracellular signal-related (ERK) mitogen-activated protein kinases (MAPKs), and activated ERK MAPKs was determined by immunoblotting. Claudin membrane association was assessed by immunohistochemistry and claudin messenger RNA expression by Northern blot analysis. Intestinal epithelial barrier function was characterized through transepithelial electrical resistance and mannitol tracer flux. RESULTS:IL-17 induced the development of a paracellular barrier of T84 cell monolayers. Inhibition of ERK activation with the MEK inhibitor PD98059 blocked IL-17 as well as basal development of tight junctions in T84 cells. IL-17 induced formation of tight junctions correlated with up-regulation of claudin-1 and claudin-2 gene transcription. Inhibition of MEK reduced the activated and basal expression of claudin-2 messenger RNA and protein expression. Functional MEK was required for the expression and membrane association of claudin-2 but not claudin-1 in T84 cells. CONCLUSIONS:MEK activity is required for claudin-mediated formation of tight junctions. IL-17 is able to regulate the intestinal barrier through the ERK MAPK pathway.
Involvement of claudin-1 in the beta-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers.
Miwa N,Furuse M,Tsukita S,Niikawa N,Nakamura Y,Furukawa Y
Accumulation of beta-catenin in cytoplasm and nuclei is frequently observed in a wide variety of tumors arising, for example, in the colon, liver, uterus, or brain. In association with Tcf/LEF transcription factors, beta-catenin regulates expression of genes involved in the Wnt/wingless signaling pathway, but the precise mechanisms are unclear. Here we report evidence that the claudin-1 (CLDNI) gene is one of the genes regulated by beta-catenin. Not only did expression of CLDN1 decrease significantly in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of wild-type APC into the APC-deficient colon cancer cells, but also two putative Tcf4 binding elements in the 5' flanking region of CLDN1 were confirmed to be responsible for activating its transcription. We documented increased expression of CLDN1 in all 16 primary colorectal cancers we examined, compared with adjacent noncancerous mucosae. Furthermore, immunohistochemical staining demonstrated that claudin-1 was weakly stained at apical boarder of lateral membrane of noncancerous epithelial cells and that it was strongly stained at all cell-cell boundaries and in the cytoplasms of cancer cells. Our results imply that claudin-1 is involved in the beta-catenin-Tcf/LEF signaling pathway, and that increased expression of claudin-1 may have some role in colorectal tumorigenesis.
Claudin-1 overexpression in intestinal epithelial cells enhances susceptibility to adenamatous polyposis coli-mediated colon tumorigenesis.
Pope Jillian L,Ahmad Rizwan,Bhat Ajaz A,Washington Mary K,Singh Amar B,Dhawan Punita
BACKGROUND:The tight junction protein Claudin-1, a claudin family member, has been implicated in several gastro-intestinal pathologies including inflammatory bowel disease (IBD) and colorectal cancer (CRC). In this regard, we have demonstrated that claudin-1 expression in colon cancer cells potentiates their tumorigenic ability while in vivo expression of claudin-1 in the intestinal epithelial cells (IECs) promotes Notch-activation, inhibits goblet cell differentiation and renders susceptibility to mucosal inflammation. Notably, a key role of inflammation in colon cancer progression is being appreciated. Therefore, we examined whether inflammation plays an important role in claudin-1-dependent upregulation of colon carcinogenesis. METHODS:APCmin mice were crossed with Villin-claudin-1 transgenic mice to generate APC-Cldn1 mice. H&E stained colon tissues were assessed for tumor number, size and histological grade. Additionally, microarray and qPCR analyses of colonic tumors were performed to assess molecular changes due to claudin-1 expression. APC-Cldn1 and APCmin controls were assessed for colonic permeability via rectal administration of FITC-dextran, and bacterial translocation via qPCR analysis of 16S rDNA. RESULTS:Claudin-1 overexpression in APCmin mice significantly increased (~4-fold) colonic tumor growth and size, and decreased survival. Furthermore, transcriptome analysis supported upregulated proliferation, and increased Wnt and Notch-signaling in APC-Cldn1 mice. APC-Cldn1 mice also demonstrated inhibition of mucosal defense genes while expression of pro-inflammatory genes was sharply upregulated, especially the IL-23/IL-17 signaling. We predict that increased Notch/Wnt-signaling underlie the early onset of adenoma formation in APC-Cldn1 mice. An increase in mucosal permeability due to the adenomas and the inherent barrier defect in these mice further facilitate bacterial translocation into the mucosa to induce inflammation, which in turn promote the tumorigenesis. CONCLUSION:Taken together, these results confirm the role of claudin-1 as a promoter of colon tumorigenesis and further identify the role of the dysregulated antigen-tumor interaction and inflammation in claudin-1-dependent upregulation of colon tumorigenesis.
Polymorphisms in the CLDN1 and CLDN7 genes are related to differentiation and tumor stage in colon carcinoma.
Hahn-Strömberg Victoria,Askari Shlear,Befekadu Rahel,Matthiessen Peter,Karlsson Sune,Nilsson Torbjörn K
APMIS : acta pathologica, microbiologica, et immunologica Scandinavica
Tight junction is composed of transmembrane proteins important for maintaining cell polarity and regulating ion flow. Among these proteins are the tissue-specific claudins, proteins that have recently been suggested as tumor markers for several different types of cancer. An altered claudin expression has been observed in colon, prostatic, ovarian, and breast carcinoma. The aim of this study was to analyze the allele frequencies of three common single nucleotide polymorphisms (SNPs) in the genes for claudin 1 and claudin 7 in colon cancer (CC) patients and in a control population of healthy blood donors. Pyrosequencing was used to genotype the CLDN1 SNP rs9869263 (c.369C>T), and the CLDN7 SNPs rs4562 (c.590C>T) and rs374400 (c.606T>G) in DNA from 102 formalin fixed paraffin embedded (FFPE) colon cancer tissue, and 111 blood leukocyte DNA from blood/plasma donors. These results were correlated with clinical parameters such as TNM stage, tumor localization, tumor differentiation, complexity index, sex, and age. We found that there was a significant association between the CLDN1 genotype CC in tumor samples and a higher risk of colon cancer development (OR 3.0, p < 0.001). We also found that the CLDN7 rs4562 (c.590C>T) genotype CT had a higher risk of lymph node involvement (p = 0.031) and a lower degree of tumor differentiation (p = 0.028). In the control population, the allele frequencies were very similar to those in the HapMap cohort for CLDN7. The CLDN1 rs9869263 genotype (c.369C>T) was related to increased risk of colon cancer, and the CLDN7 rs4562 genotype (c.590C>T) was related to tumor differentiation and lymph node involvement in colon carcinoma. Further studies are warranted to ascertain their potential uses as biomarkers predicting tumor development, proliferation, and outcome in this disease.
Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.
Ding Lei,Wang Liyong,Sui Leiming,Zhao Huanying,Xu Xiaoxue,Li Tengyan,Wang Xiaonan,Li Wenjing,Zhou Ping,Kong Lu
Journal of human genetics
The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.
Claudin expression in rectal well-differentiated endocrine neoplasms (carcinoid tumors).
Ishida Mitsuaki,Kushima Ryoji,Okabe Hidetoshi
Claudins are the structures and functional components of tight junctions and have crucial roles in the maintenance of cell polarity, cellular arrangement, adhesion and paracellular transport. Various claudins are expressed in different epithelial cells and most tissues express multiple claudin proteins. The altered expression of claudins has been reported in various human carcinomas, but their expression in rectal well-differentiated endocrine neoplasms (carcinoid tumors), the most common endocrine tumors in the gastrointestinal tract, has yet to be examined. The expression of claudins-2, -3 and -4 in 16 rectal well-differentiated endocrine neoplasms was studied by immunohistochemical methods, and compared with their expression patterns in endocrine tumors of the pancreas and lung. According to previous reports, pancreatic endocrine tumors were positive for claudin-3 and negative for claudins-2 and -4, and a majority of lung carcinoid tumors showed no immunoreactivity to claudins-2, -3 and -4. However, our immunohistochemical study revealed that the rectal well-differentiated endocrine neoplasms showed diffuse positive immunoreactivity to claudins-2, -3 and -4. These results indicate that claudin expression depends on the site of origin of endocrine tumors. In addition, claudin-3 and -4 expression in rectal well-differentiated endocrine neoplasms suggests the possibility of a new therapeutic strategy. Claudins-3 and -4 are receptors for the cytotoxic Clostridium perfringens enterotoxin. This enterotoxin rapidly and specifically lyses cells expressing claudins-3 and -4 and has a potential application in cancer therapeutics. Accordingly, this enterotoxin may be applicable for the treatment of rectal well-differentiated endocrine neoplasms in the future in order to prevent unexpected metastatic recurrences after tumor resections, because these neoplasms have a relatively high incidence of metastases despite their small size.
Claudin-1 expression confers resistance to anoikis in colon cancer cells in a Src-dependent manner.
Singh Amar B,Sharma Ashok,Dhawan Punita
Denial of the appropriate cell-matrix interaction in epithelial cells induces apoptosis and is called 'anoikis'. Cancer cells are resistant to anoikis and it is believed that the resistance to anoikis helps promote tumor malignancy especially metastasis. We and others have demonstrated that the expression of tight junction protein claudin-1 is highly upregulated in colorectal cancer (CRC) and helps promote tumor progression and metastasis. However, molecular mechanism/s underlying claudin-1-dependent regulation of CRC progression remains poorly understood. In current study, we have determined that claudin-1 expression modulates anoikis in colon cancer cells to influence colon cancer invasion and thus metastasis. We have further provided data that claudin-1 modulates anoikis in a Src-Akt-Bcl-2-dependent manner. Importantly, claudin-1 physically associates with Src/p-Src in a multiprotein complex that also includes ZO-1, a PDZ-binding tight junction protein. Taken together, our data support the role of claudin-1 in the regulation of CRC progression and suggest that the regulation of anoikis may serve as a key regulatory mechanism in claudin-1-dependent regulation of CRC progression. Our findings are of direct clinical relevance and may open new therapeutic opportunity in colon cancer treatment and/or management.
Claudin-1 up-regulates the repressor ZEB-1 to inhibit E-cadherin expression in colon cancer cells.
Singh Amar B,Sharma Ashok,Smith J Joshua,Krishnan Moorthy,Chen Xi,Eschrich Steven,Washington Mary K,Yeatman Timothy J,Beauchamp R Daniel,Dhawan Punita
BACKGROUND & AIMS:Expression of the tight junction protein claudin-1 is dysregulated in colon tumors and associates with their progression. Up-regulation of claudin-1 reduces expression of E-cadherin. We investigated the mechanisms by which claudin-1 regulates E-cadherin expression and its effects in colon cancer cells. MATERIALS AND METHODS:We used gene expression analysis, immunoblotting, and reverse transcription polymerase chain reaction to associate expression of the repressor of transcription Zinc Finger E-box binding homeobox-box1 (ZEB-1) with claudin-1. We analyzed SW480 colon cancer cells that overexpressed claudin-1, or SW620 cells in which claudin-1 expression was repressed, to determine the effects on ZEB-1 and E-cadherin expression, invasive activity, and resistance to anoikis. We studied cells that expressed constitutively active or dominant negative forms of factors in the Wnt or phosphotidylinositol-3-kinase signaling pathways and used pharmacologic inhibitors of these pathways to study their role in claudin-1-dependent regulation of ZEB-1. We used microarray analysis to examine gene expression patterns in 260 colorectal tumor and normal colon samples. RESULTS:Claudin-1 down-regulates E-cadherin expression by up-regulating expression of ZEB-1. Claudin-1 activates Wnt and phosphotidylinositol-3-kinase/Akt signaling. ZEB-1 mediates claudin-1-regulated changes in cell invasion and anoikis. Expression of claudin-1 correlated with that of ZEB-1 in human colon tumor samples. In the progression from normal colonic epithelium to colon adenocarcinoma, levels of E-cadherin decreased, whereas levels of claudin-1 and ZEB-1 increased. Down-regulation of E-cadherin and up-regulation of ZEB-1 in colon tumors were associated with shorter survival times. CONCLUSIONS:Claudin-1 up-regulates the repressor ZEB-1 to reduce expression of E-cadherin in colon cancer cells, increasing their invasive activity and reducing anoikis. This pathway is associated with colorectal cancer progression and patient survival.
Expression of tight and adherens junction proteins in ulcerative colitis associated colorectal carcinoma: upregulation of claudin-1, claudin-3, claudin-4, and beta-catenin.
Mees S T,Mennigen R,Spieker T,Rijcken E,Senninger N,Haier J,Bruewer M
International journal of colorectal disease
BACKGROUND:Tight junction (TJ) proteins play a critical role in cellular adhesion, glandular differentiation, and cellular proliferation. The function of these proteins is compromised in a number of intestinal diseases, including ulcerative colitis that has an increased incidence for colorectal carcinoma (CAC). The aim of this study was to determine the expression of TJ proteins, claudin-1-4, occludin, ZO-1, and the adherens junction (AJ) protein beta-catenin in CAC. METHODS:Sixteen colectomy specimens with CAC, adjoining intraepithelial neoplasia, and normal mucosa were studied by immunofluorescence. A semiquantitative evaluation of all investigated proteins was performed by scoring the staining intensity, and the TJ and AJ protein expression in neoplastic cells was compared to normal and intraepithelial neoplastic colonic mucosa. RESULTS:Using an intensity scoring system, mucosa of crypts and surfaces of CAC exhibited significantly elevated expression levels of claudin-1, claudin-3, claudin-4, and beta-catenin compared to intraepithelial neoplasia and normal mucosa (p<0.05). These data were confirmed by a comparative score. The expression of claudin-2, occludin, and ZO-1 showed no differences between the groups. CONCLUSION:TJ proteins claudin-1, claudin-3, claudin-4, and the AJ protein beta-catenin are overexpressed in CAC. This suggests that these proteins may become potential markers and targets in CAC.
Caudal homeobox protein Cdx-2 cooperates with Wnt pathway to regulate claudin-1 expression in colon cancer cells.
Bhat Ajaz A,Sharma Ashok,Pope Jillian,Krishnan Moorthy,Washington Mary K,Singh Amar B,Dhawan Punita
Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5'-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated β-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.
Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.
Tokuhara Yasunori,Morinishi Tatsuya,Matsunaga Toru,Sakai Manabu,Sakai Takayoshi,Ohsaki Hiroyuki,Kadota Kyuichi,Kushida Yoshio,Haba Reiji,Hirakawa Eiichiro
Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in detail, the present results indicated the potential of claudin-3 as a histopathological biomarker for colorectal adenocarcinomas.
[The expression of claudins in colonic neoplasms].
Oleinikova N A,Kharlova O A,Mal'kov P G,Danilova N V
AIM:the evaluation of localization claudin-1, -3 and -4 types of cancer and colon polyps. MATERIAL AND METHODS:The study included 32 colon adenocarcinoma and 86 polyps. Antibody claudin-1; -3 and -4 were used as immunohistochemical markers in this study. RESULTS:84/118, 64/118, 52/118 reaction with claudin-1, claudin-3 and claudin-4 in cancer and colon polyps had a membrane localization, respectively. In 33 (27.9%) cases was found paradoxical reaction claudin-1; in 50 (42.4%) - a paradoxical reaction claudin-3 in 66 (55.9%) - a paradoxical reaction claudin-4. Among the paradoxical claudin reaction nuclear localization of marker was observed relatively rarely: claudin-3 in 2.5% cases of colon cancer; claudin-4 in 8.5% of colon polyps. CONCLUSION:Mislocalization claudin-3 to nucleus in colon cancer and mislocalization claudin-4 to nucleus in adenomas of the colon were detected for the first time. The potential reasons for the paradoxical expression are discussed and a review of the literature, related all the alleged mechanisms of this mislocalization is provided.
Differential expression of claudin-2 in normal human tissues and gastrointestinal carcinomas.
Aung Phyu Phyu,Mitani Yoshitsugu,Sanada Yuichi,Nakayama Hirofumi,Matsusaki Keisuke,Yasui Wataru
Virchows Archiv : an international journal of pathology
Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. Claudins form a family of 24 members displaying organ- and tissue-specific patterns of expression. In the present study, we evaluated the specificity of the claudin-2 expression in various normal human tissues and gastrointestinal cancers by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. In 14 various normal tissues, claudin-2 mRNA was expressed in the kidney, liver, pancreas, stomach, and small intestine; the highest level of which was detected in the kidney. Colorectal cancers (CRCs) expressed claudin-2 mRNA at high levels. Immunohistochemical analysis of claudin-2 in 146 gastric cancers (GCs) and 99 CRCs demonstrated claudin-2 expression in 2.1% of GCs and 25.3% of CRCs, respectively. There was no obvious correlation between claudin-2 expression and clinicopathological parameters of CRCs. These results suggest that the expression of claudin-2 may involve organ specificity, and increased expression of claudin-2 may participate in colorectal carcinogenesis.
Investigation of Metastasis-Related Genes: A Rat Model Mimicking Liver Metastasis of Colorectal Carcinoma.
Adwan Hassan,Georges Rania,Pervaiz Asim,Berger Martin R
Frontiers in oncology
Liver is the main target of colorectal cancer (CRC) metastasis. Currently, the number of reports is small, which describe changes in gene expression supporting liver metastasis. Here, a rat model was used for analyzing mRNA modulations during liver colonization and compared with available literature. In the model, CC531 rat CRC cells were injected a mesenteric vein into isogenic WAG/Rij rats and re-isolated at early, intermediate, advanced, and terminal stages of liver colonization. These cells were used for RNA isolation. Microarrays were used for analyzing mRNA profiles of expression. The number of deregulated genes is comparatively large and only part of it has been studied so far. As reported to date, claudins and insulin-like growth factor-binding proteins (IGFBPs) were found to be deregulated. The fact that the chosen method is efficient is confirmed by the study of claudins and IGFBPs, which show altered expression in the initial stages of liver colonization and then return to normalcy. In addition, cadherin was described to be downregulated in epithelial-mesenchymal transition models. It can, therefore, be concluded that the models used are helpful in finding genes, which are instrumental for metastatic liver colonization.
Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids.
Bardenbacher Marco,Ruder Barbara,Britzen-Laurent Nathalie,Schmid Benjamin,Waldner Maximilian,Naschberger Elisabeth,Scharl Michael,Müller Werner,Günther Claudia,Becker Christoph,Stürzl Michael,Tripal Philipp
Stem cell research
The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However, established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-γ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-γ-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-γ-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2, -7, and - 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary, we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model.
Cluster analysis of claudin-1 and -4, E-cadherin, and β-catenin expression in colorectal cancers.
Matsuoka Takashi,Mitomi Hiroyuki,Fukui Naoshi,Kanazawa Hideki,Saito Tsuyoshi,Hayashi Takuo,Yao Takashi
Journal of surgical oncology
BACKGROUND AND OBJECTIVES:Intercellular adhesion mediated by the claudin and cadherin/catenin complex is a prerequisite for epithelial integrity and differentiation, and has been suggested to be frequently disturbed in cancers. The aim of this study was to assess the relationship between such abnormality and clinicopathological features of colorectal carcinomas. METHODS:Immunohistochemical analysis of claudin-1 and -4, E-cadherin, and β-catenin was performed on a series of 156 cases. RESULTS:Significant positive associations (P < 0.05-0.001) were found with immunoreactivity for each except nuclear β-catenin. Reduced expression was correlated with poor tumor differentiation (claudin-1, P = 0.035; claudin-4, P = 0.011; E-cadherin, P < 0.001; membranous β-catenin, P = 0.002), an advanced TNM stage (claudin-1, P = 0.002; claudin-4, P = 0.008), and a poor prognosis. On multivariate analysis, reduced expression of E-cadherin (P < 0.001) and β-catenin (P = 0.048) at invasive fronts proved to be an independent predictor of short survival. Hierarchical cluster analysis identified three distinct groups with a good, intermediate, and poor prognosis, having an independent survival outcome by multivariate analysis (good or intermediate vs. poor: hazard ratio, 2.66; 95% confidence interval, 1.54-4.60; P < 0.001). CONCLUSIONS:Disruption of cell adhesion molecules correlates with tumor differentiation and progression in colorectal carcinomas. Specific marker profiles were identified here as independent prognostic indicators.
Hypermethylation-modulated downregulation of claudin-7 expression promotes the progression of colorectal carcinoma.
Nakayama Fumihito,Semba Shuho,Usami Yu,Chiba Hideki,Sawada Norimasa,Yokozaki Hiroshi
Pathobiology : journal of immunopathology, molecular and cellular biology
OBJECTIVE:The expression of tight junction-related transmembrane protein claudin-7 (CLDN7) and its regulatory mechanism were investigated in colorectal carcinomas (CRCs). METHODS:Methylation-specific polymerase chain reaction and treatment with the demethylating agent 5-aza-2'-deoxycytidine were conducted to analyze the methylation status at the CLDN7 promoter region in the Colo320 CRC cell line. We used a total of 26 stage 0 CRCs with an adenoma component and 90 invasive CRCs (stage I-IV), as well as their corresponding lymph node metastases, in an immunohistochemical study. RESULTS:In Colo320 (CLDN7-negative) cells, hypermethylation at the CLDN7 promoter was detected and treatment with 5-aza-2'-deoxycytidine restored CLDN7 expression. In CRC tissues, decreased CLDN7 expression was detected in 62% of stage 0 CRCs and 80% of stage I-IV CRCs, compared with their adjacent adenoma lesions and nonneoplastic epithelia, which had a close correlation with the incidence of vessel infiltration and clinicopathologic stage. Hypermethylation at the CLDN7 promoter was detected in 20% of CRCs with low CLDN7 expression. However, CLDN7 expression tended to be re-expressed in their corresponding lymph node metastases. CONCLUSION:These findings suggest that the CLDN7 gene silencing by promoter hypermethylation and the resultant reduction of CLDN7 expression may play an important role in the progression of CRCs.
Smad4 regulates claudin-1 expression in a transforming growth factor-beta-independent manner in colon cancer cells.
Shiou Sheng-Ru,Singh Amar B,Moorthy Krishnan,Datta Pran K,Washington M Kay,Beauchamp R Daniel,Dhawan Punita
We have recently reported that the expression of a tight junction protein, claudin-1, is increased during colon carcinogenesis and particularly metastatic colorectal cancer. Manipulation of claudin-1 levels in colon cancer cells showed a positive correlation between claudin-1 expression and tumor growth and metastasis. However, the mechanisms underlying the increased claudin-1 expression in colorectal cancer remains unknown. The tumor suppressor Smad4 is a central intracellular signal transduction component of the transforming growth factor-beta (TGF-beta) family of cytokines. Loss of Smad4 protein expression is correlated with poor prognosis and is frequently observed in invasive and metastatic colorectal carcinoma. In the present study, we report an inverse relationship between Smad4 and claudin-1 expression in human colorectal carcinoma tumor samples and in human colon cancer cell lines. We found that the expression of Smad4 in Smad4-deficient but claudin-1-positive SW480 or HT29 colon cancer cell lines down-regulates claudin-1 expression through transcriptional repression by modulating beta-catenin/T-cell factor/lymphocyte enhancer factor activity. Furthermore, this Smad4-dependent inhibition of claudin-1 expression is independent of TGF-beta signaling because Smad4 expression alone is insufficient to restore TGF-beta signaling in the SW480 cells, and the selective TGF-beta receptor kinase inhibitor LY364947 did not prevent the Smad4 suppression of claudin-1 protein expression in either SW480 or HT29 cells. Taken together, these findings suggest a novel mechanism underlying Smad4 tumor-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner.
Immunohistochemical staining of Reg IV and claudin-18 is useful in the diagnosis of gastrointestinal signet ring cell carcinoma.
Sentani Kazuhiro,Oue Naohide,Tashiro Takashi,Sakamoto Naoya,Nishisaka Takashi,Fukuhara Toshiyuki,Taniyama Kiyomi,Matsuura Hiroo,Arihiro Koji,Ochiai Atsushi,Yasui Wataru
The American journal of surgical pathology
Signet-ring cell carcinoma (SRCC) is a unique subtype of adenocarcinoma that is characterized by abundant intracellular mucin accumulation and a crescent-shaped nucleus displaced toward one end of the cell. Identification of an SRCC's primary site is important for better planning of patient management because the treatment and prognosis differs markedly depending on the origin of the SRCC. In the present study, we analyzed the immunohistochemical characteristics of 94 cases of SRCC, including 21 cases of gastric SRCC, 16 of colorectal SRCC, 10 of breast SRCC, and 47 of pulmonary SRCC, with antibodies against Reg IV and claudin-18, which we previously identified as gastric cancer-related genes. We also tested known markers cytokeratin 7, cytokeratin 20, MUC2, MUC5AC, caudal-related homeobox gene 2 (CDX2), thyroid transcription factor-1, mammaglobin, gross cystic disease fluid protein 15, and estrogen receptor. All 21 cases of gastric SRCC and 16 cases of colorectal SRCC were positive for Reg IV, and the remaining SRCCs were negative. Eighteen of 21 (86%) gastric SRCCs and 6 of 16 (38%) colorectal SRCCs were positive for claudin-18, whereas another SRCCs were negative. In conclusion, Reg IV staining and claudin-18 staining can aid in diagnosis of gastrointestinal SRCC.
Severe Intestinal Inflammation in the Small Intestine of Mice Induced by Controllable Deletion of Claudin-7.
Li Wen-Jing,Xu Chang,Wang Kun,Li Teng-Yan,Wang Xiao-Nan,Yang Hui,Xing Tiaosi,Li Wen-Xia,Chen Yan-Hua,Gao Hong,Ding Lei
Digestive diseases and sciences
BACKGROUND:As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development. AIMS:To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen. METHODS:We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis. RESULTS:After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened. CONCLUSIONS:We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice.
Identification of putative immunologic targets for colon cancer prevention based on conserved gene upregulation from preinvasive to malignant lesions.
Broussard Elizabeth K,Kim Rachel,Wiley Jesse C,Marquez Juan Pablo,Annis James E,Pritchard David,Disis Mary L
Cancer prevention research (Philadelphia, Pa.)
The length of time required for preinvasive adenoma to progress to carcinoma, the immunogenicity of colorectal cancer (CRC), and the identification of high-risk populations make development and testing of a prophylactic vaccine for the prevention of CRC possible. We hypothesized that genes upregulated in adenoma relative to normal tissue, which maintained increased expression in CRC, would encode proteins suitable as putative targets for immunoprevention. We evaluated existing adenoma and CRC microarray datasets and identified 160 genes that were ≥2-fold upregulated in both adenoma and CRC relative to normal colon tissue. We further identified 23 genes that showed protein overexpression in colon adenoma and CRC based on literature review. Silencing the most highly upregulated genes, CDH3, CLDN1, KRT23, and MMP7, in adenoma and CRC cell lines resulted in a significant decrease in viability (P < 0.0001) and proliferation (P < 0.0001) as compared to controls and an increase in cellular apoptosis (P < 0.05 for CDH3, KRT23). Results were duplicated across cell lines representing microsatellite instability, CpG island methylator, and chromosomal instability phenotypes, suggesting immunologic elimination of cells expressing these proteins could impact the progression of all CRC phenotypes. To determine whether these proteins were immunogens, we interrogated sera from early stage CRC patients and controls and found significantly elevated CDH3 (P = 0.006), KRT23 (P = 0.0007), and MMP7 (P < 0.0001) serum immunoglobulin G in cases as compared to controls. These data show a high throughput approach to the identification of biologically relevant putative immunologic targets for CRC and identified three candidates suitable for vaccine development.
Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.
Bhat A A,Pope J L,Smith J J,Ahmad R,Chen X,Washington M K,Beauchamp R D,Singh A B,Dhawan P
In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.
[Study of claudins and prognostic factors in some gastrointestinal diseases].
Gastrointestinal tumors are highly ranked regarding tumoral mortality worldwide. The development and progression of gastrointestinal (GI) diseases go hand in hand with the changes of tight junctions (TJ). Claudins (CLDN) are the main TJ proteins, showing different expression by the different tissues, with the expressed CLDN profile being representative. I. We explored the changes of CLDN expression in Barrett's esophagus and related adenocarcinoma. CLDN2 and -3 expression in Barrett's esophagus was higher than in normal foveolar epithelium. Adenocarcinoma showed higher CLDN2 and -3 expression compared with normal and Barrett's epithelia. The similar CLDN expression profile of Barrett's esophagus and adenocarcinoma supports their sequential development. II. Gastric intestinal metaplasia showed higher expression of CLDN2, -3 and -4 as compared with normal antral foveolar mucosa. Tumors of small and large bowels exhibited higher CLDN2 expression when compared with normal epithelia. Colorectal adenoma and adenocarcinoma could not be differentiated according to their CLDN profile. Intestinal metaplasias of Barrett's esophagus and stomach show similar CLDN profile to small bowel epithelium. III. Studies on duodenal mucosa in celiac disease in childhood demonstrated CLDN2 and -3 expression to be higher than in normal mucosa. The expression was significantly higher in the distal part of the duodenum samples. This and the serious histological findings suggest that the distal duodenum is more adequate for biopsy testing. IV. Beside the epithelial cells, mesenchymal tumors express intercellular junctional proteins. Expression of claudins in gastrointestinal stromal tumors (GIST) and other mesenchymal neoplasia was also studied. The CLDN profile was found to be representative to the individual tumor. GIST, angiosarcoma, hemangioma, leiomyosarcoma and leiomyoma showed expression of various CLDNs. CLDN2 was detected in all entities. CLDN1, however, was found positive in leiomyosarcoma only. Leiomyoma, on the other hand, expressed only CLDN2. GISTs and leiomyosarcomas showed CLDN2, -3, -4, -5 and -7-expression. The angiogenic tumors revealed CLDN2 and -5 expression. The similar CLDN profile observable in GIST and leiomyosarcoma is suggestive of a histogenetic relationship. Smooth muscle and vessel tumors of different dignity could also be separated from each other based on CLDN profile.
Claudin-2 expression increases tumorigenicity of colon cancer cells: role of epidermal growth factor receptor activation.
Dhawan P,Ahmad R,Chaturvedi R,Smith J J,Midha R,Mittal M K,Krishnan M,Chen X,Eschrich S,Yeatman T J,Harris R C,Washington M K,Wilson K T,Beauchamp R D,Singh A B
Claudin-2 is a unique member of the claudin family of transmembrane proteins, as its expression is restricted to the leaky epithelium in vivo and correlates with epithelial leakiness in vitro. However, recent evidence suggests potential functions of claudin-2 that are relevant to neoplastic transformation and growth. In accordance, here we report, on the basis of analysis of mRNA and protein expression using a total of 309 patient samples that claudin-2 expression is significantly increased in colorectal cancer and correlates with cancer progression. We also report similar increases in claudin-2 expression in inflammatory bowel disease-associated colorectal cancer. Most importantly, we demonstrate that the increased claudin-2 expression in colorectal cancer is causally associated with tumor growth as forced claudin-2 expression in colon cancer cells that do not express claudin-2 resulted in significant increases in cell proliferation, anchorage-independent growth and tumor growth in vivo. We further show that the colonic microenvironment regulates claudin-2 expression in a manner dependent on signaling through the EGF receptor (EGFR), a key regulator of colon tumorigenesis. In addition, claudin-2 expression is specifically decreased in the colon of waved-2 mice, naturally deficient in EGFR activation. Furthermore, genetic silencing of claudin-2 expression in Caco-2, a colon cancer cell line, prevents the EGF-induced increase in cell proliferation. Taken together, these results uncover a novel role for claudin-2 in promoting colon cancer, potentially via EGFR transactivation.
Symplekin promotes tumorigenicity by up-regulating claudin-2 expression.
Buchert Michael,Papin Marina,Bonnans Caroline,Darido Charbel,Raye Warren S,Garambois Véronique,Pélegrin André,Bourgaux Jean-François,Pannequin Julie,Joubert Dominique,Hollande Frédéric
Proceedings of the National Academy of Sciences of the United States of America
Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Analysis of Snail-1, E-cadherin and claudin-1 expression in colorectal adenomas and carcinomas.
International journal of molecular sciences
We report the expression of Snail-1, E-cadherin and claudin-1 by indirect immunohistochemistry in colonic neoplasia. Snail-1 is a zinc finger transcription factor expressed in cells that already have undergone almost complete epithelial-mesenchymal transition (EMT) and have already evaded from the tumor. The main mechanism by which Snail induces EMT is downregulation of E-cadherin, of which expression was shown to be frequently downregulated in many different types of tumors, where it accompanies the invasiveness and metastatic behavior of malignant cells. Moreover, Snail-1 may downregulate the expression of claudin-1, a cell-cell adhesion protein which plays a likely role in progression and dissemination during tumorigenesis. Snail-1 was expressed in both carcinoma and adenoma cells with histologically normal epithelium in the mucosa, adjacent to the tumors, without significant differences, and predominant strong intensity of staining. Statistically significant differences were revealed between normal and tumorous epithelium (p = 0.003) at the subcellular level, where the shift of the protein to the cytoplasm with combined cytoplasmic/nuclear or pure cytoplasmic expression was observed. E-cadherin expression was present in 100% of cases of both adenocarcinomas and adenomas, with prevailing strong membranous immunoreactivity and no differences between protein expression in tumors and normal mucosa. Predominating strong positivity of claudin-1 was detected in tumor cells of adenocarcinomas and adenomas. Marked differences were seen in protein localization, where membranous staining, typical for nontumorous epithelium, changed to combined membranous/cytoplasmic expression in adenocarcinomas (p = 0.0001) and adenomas (0.0002), in which cytoplasmic shift was associated with a higher degree of dysplasia. Furthermore, membranous/cytoplasmic localization was more frequent in the carcinoma group (87%) in comparison with adenomas (51%) (p = 0.0001). We conclude that dystopic subcellular localizations of Snail-1 and claudin-1 may participate in changes of cellular morphology and behavior which might be associated with altered effectory pathways of proteins and thus substantially contribute to the cancer development.
Lyn modulates Claudin-2 expression and is a therapeutic target for breast cancer liver metastasis.
Tabariès Sébastien,Annis Matthew G,Hsu Brian E,Tam Christine E,Savage Paul,Park Morag,Siegel Peter M
Claudin-2 enhances breast cancer liver metastasis and promotes the development of colorectal cancers. The objective of our current study is to define the regulatory mechanisms controlling Claudin-2 expression in breast cancer cells. We evaluated the effect of several Src Family Kinase (SFK) inhibitors or knockdown of individual SFK members on Claudin-2 expression in breast cancer cells. We also assessed the potential effects of pan-SFK and SFK-selective inhibitors on the formation of breast cancer liver metastases. This study reveals that pan inhibition of SFK signaling pathways significantly elevated Claudin-2 expression levels in breast cancer cells. In addition, our data demonstrate that pan-SFK inhibitors can enhance breast cancer metastasis to the liver. Knockdown of individual SFK members reveals that loss of Yes or Fyn induces Claudin-2 expression; whereas, diminished Lyn levels impairs Claudin-2 expression in breast cancer cells. The Lyn-selective kinase inhibitor, Bafetinib (INNO-406), acts to reduce Claudin-2 expression and suppress breast cancer liver metastasis. Our findings may have major clinical implications and advise against the treatment of breast cancer patients with broad-acting SFK inhibitors and support the use of Lyn-specific inhibitors.
Expression of olfactomedin 4 and claudin-18 in serrated neoplasia of the colorectum: a characteristic pattern is associated with sessile serrated lesion.
Sentani Kazuhiro,Sakamoto Naoya,Shimamoto Fumio,Anami Katsuhiro,Oue Naohide,Yasui Wataru
AIMS:Olfactomedin 4 is a useful marker for stem cells in the intestine and is an independent prognostic molecule for survival in patients with colorectal cancer (CRC). Claudin-18, a component of tight junctions, correlates with poor survival in patients with CRC and is associated with the gastric phenotype. We investigated the possible usefulness of these molecules in serrated neoplasia of the colorectum. METHODS AND RESULTS:We performed immunohistochemical analysis of colorectal polyps, including hyperplastic polyps (HP), sessile serrated lesions (SSL), traditional serrated adenomas (TSA) and conventional adenomas (CA). We also investigated the association between expression of these molecules and clinicopathological parameters in serrated adenocarcinoma (SAC) and non-SAC of the colorectum. Olfactomedin 4 expression was not detected or was decreased in SSL compared with the other polyp types. Claudin-18 expression was higher in SSL than in the other types. Similarly, positivity for olfactomedin 4 in SAC was significantly lower than that in non-SAC, and positivity for claudin-18 in SAC was significantly higher than that in non-SAC. Furthermore, claudin-18-positive SAC showed more advanced N grade and stage than claudin-18-negative SAC. CONCLUSIONS:Reduced expression of olfactomedin 4 and ectopic expression of claudin-18 might be useful markers in the differential diagnosis of serrated polyps.
Collective migration of cancer-associated fibroblasts is enhanced by overexpression of tight junction-associated proteins claudin-11 and occludin.
Karagiannis George S,Schaeffer David F,Cho Chan-Kyung J,Musrap Natasha,Saraon Punit,Batruch Ihor,Grin Andrea,Mitrovic Bojana,Kirsch Richard,Riddell Robert H,Diamandis Eleftherios P
It has been suggested that cancer-associated fibroblasts (CAFs) positioned at the desmoplastic areas of various types of cancer are capable of executing a migratory program, characterized by accelerated motility and collective configuration. Since CAFs are reprogrammed derivatives of normal progenitors, including quiescent fibroblasts, we hypothesized that such migratory program could be context-dependent, thus being regulated by specific paracrine signals from the adjacent cancer population. Using the traditional scratch assay setup, we showed that only specific colon cancer cell lines (i.e. HT29) were able to induce collective CAF migration. By performing quantitative proteomics (SILAC), we identified a 2.7-fold increase of claudin-11, a member of the tight junction apparatus, in CAFs that exerted such collectivity in their migratory pattern. Further proteomic investigations of cancer cell line secretomes revealed a specific signature, involving TGF-β, as potential mediator of this effect. Normal colonic fibroblasts stimulated with TGF-β exerted myofibroblastic differentiation, occludin (OCLN) and claudin-11 (CLDN11) overexpression and cohort formation. Subsequently, inhibition of TGF-β attenuated all the previous effects. Immunohistochemistry of the universal tight junction marker occludin in a cohort of 30 colorectal adenocarcinoma patients defined a CAF subpopulation expressing tight junctions. Overall, these data suggest that cancer cells may induce CLDN11 overexpression and subsequent collective migration of peritumoral CAFs via TGF-β secretion.
Prognostic significance of the genetic and the immunohistochemical expression of epithelial-mesenchymal-related markers in colon cancer.
Rashed Hayam E,Hussein Samia,Mosaad Hala,Abdelwahab Mai M,Abdelhamid Mohamed I,Mohamed Salem Y,Mohamed Abdel Motaleb,Fayed Alaa
Cancer biomarkers : section A of Disease markers
BACKGROUND:Epithelial-mesenchymal transition (EMT) is one of the main events in colorectal cancer (CRC) spread. Snail-1 is a zinc transcription factor that mediates EMT in tumor cells probably by down-regulation of E-cadherin and claudin-1. OBJECTIVES:To detect the expression of epithelial markers (claudin-1 and E-cadherin) and mesenchymal markers (snail-1 and vimentin) in primary cancer colon. Also, to select stage II cancer patients of a high risk that can benefit from postoperative adjuvant chemotherapy. METHODS:Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis were performed to investigate snail-1, claudin-1, E-cadherin and vimentin expressions at mRNA and protein levels in fresh tissues of cancer colon and normal colonic mucosa. The correlations between the expression of these markers and clinicopathological parameters were performed. RESULTS:Normal colonic mucosa revealed complete membranous expression of claudin-1, preserved E-cadherin and negative snail-1 and vimentin expressions. Compared to control, the expression of snail-1 and vimentin mRNA in cancer colon was significantly up-regulated while the expression of claudin-1 and E-cadherin mRNA was significantly down-regulated. These changes were significantly associated with stage and lymph node involvement at both mRNA and protein levels (p< 0.05). There were significant negative correlations between vimentin and each of E-cadherin and claudin-1 gene expression and between snail-1 and each of E-cadherin and claudin-1 gene expression. Moreover, these changes were independent predictors of recurrence of stage II cancer colon cases. CONCLUSION:There is a clinical significance of snail-1, claudin-1, E-cadherin and vimentin as possible markers for recognizing patients with lymph node involvement, advanced stage and high incidence of tumor recurrence in cancer colon.
Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy by hyperactivating Wnt/β-catenin signaling.
Ahmad R,Kumar B,Chen Z,Chen X,Müller D,Lele S M,Washington M K,Batra S K,Dhawan P,Singh A B
The hyperactivated Wnt/β-catenin signaling acts as a switch to induce epithelial to mesenchymal transition and promote colorectal cancer. However, due to its essential role in gut homeostasis, therapeutic targeting of this pathway has proven challenging. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways and key mucosal barrier components in regulating colon tumorigenesis and cancer progression remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue-specific role of claudin-3, a tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Loss of claudin-3 also predicted poor patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated role of the epigenetic regulation. Claudin-3-/- mice revealed dedifferentiated and leaky colonic epithelium, and developed invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, differentiated colon cancer in claudin-3-/- mice versus WT-mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune components. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation, which is further exacerbated by Stat-3-activation and help promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy.
Heterogeneous expression of claudin-4 in human colorectal cancer: decreased claudin-4 expression at the invasive front correlates cancer invasion and metastasis.
Ueda Junya,Semba Shuho,Chiba Hideki,Sawada Norimasa,Seo Yasushi,Kasuga Masato,Yokozaki Hiroshi
Pathobiology : journal of immunopathology, molecular and cellular biology
OBJECTIVE:Claudin-4 plays a key role in constructing the tight junction (TJ), and altered claudin-4 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin-4 in colorectal cancers (CRCs). The aim of this study is to investigate the significance of claudin-4 expression in CRC and its association with clinicopathological factors. METHODS:The levels of claudin-4 expression in a total of 129 CRCs and 44 metastatic tumors were examined by immunohistochemistry. A small interfering RNA (siRNA)-mediated claudin-4 knockdown examination was also conducted to assess the biological role(s) of claudin-4 in cultured cells. RESULTS:Expression of claudin-4 at the intercellular membrane was well preserved at the surface of the tumor; however, decreased claudin-4 expression was detected in 57% of CRCs, particularly at the invasive front. Interestingly, decreased claudin-4 expression was detected in metastatic lesions of CRC. The siRNA-mediated claudin-4 knockdown in SW480 claudin-4-positive CRC cells upregulated cell motility, whereas no significant change was detected in cell proliferation. CONCLUSIONS:These observations suggested that disruption of claudin-4-mediated TJ construction enhances cancer cell invasion and metastasis in human CRC. Claudin-4 might be a good biomarker for diagnosing the risk of distant metastasis.
Distinct claudin expression profiles of hepatocellular carcinoma and metastatic colorectal and pancreatic carcinomas.
Holczbauer Ágnes,Gyöngyösi Benedek,Lotz Gábor,Szijártó Attila,Kupcsulik Péter,Schaff Zsuzsa,Kiss András
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
Tight junction proteins, including claudins, are often dysregulated during carcinogenesis and tumor progression. Moreover, the claudin expression pattern usually varies between different tumor entities. We aimed to investigate claudin expression profiles of primary and metastatic liver malignancies. We analyzed claudin-1, -2, -3, -4, and -7 expression by quantitative immunohistochemistry and real-time RT-PCR, respectively. Twenty hepatocellular carcinomas (HCCs) and liver metastases of 20 colorectal adenocarcinomas (CRLMs) and 15 pancreatic adenocarcinomas (PLMs) were studied together with paired surrounding non-tumorous liver samples and 5 normal liver samples. Strong claudin-3 and -7 immunohistochemical positivities were detected in CRLM samples, each with significantly stronger staining when compared with HCC and PLM groups. Claudin-1 protein was found highly expressed in CRLM, in contrast to lower expression in PLM and HCC. CRLMs and PLMs also were strongly positive for claudin-4, while being virtually undetectable in HCC. Claudin-2 showed strong positivity in non-tumorous liver tissue, whereas significantly weaker positivity was observed in all tumors. Differences in mRNA expression were mostly similar to those found by immunohistochemistry. In conclusion, HCC and both CRLM and PLM display distinct claudin expression profiles, which might provide better understanding of the pathobiology of these lesions and might be used for differential diagnosis.
Claudin Family Participates in the Pathogenesis of Inflammatory Bowel Diseases and Colitis-Associated Colorectal Cancer.
Zhu Liguo,Han Jing,Li Li,Wang Ying,Li Ying,Zhang Shenghong
Frontiers in immunology
Claudins are a multigene transmembrane protein family comprising at least 27 members. In gastrointestinal tract, claudins are mainly located in the intestinal epithelia; many types of claudins form a network of strands in tight junction plaques within the intercellular space of neighboring epithelial cells and build paracellular selective channels, while others act as signaling proteins and mediates cell behaviors. Claudin dysfunction may contribute to epithelial permeation disorder and multiple intestinal diseases. Over recent years, the importance of claudins in the pathogenesis of inflammatory bowel diseases (IBD) has gained focus and is being investigated. This review analyzes the expression pattern and regulatory mechanism of claudins based on existing evidence and elucidates the fact that claudin dysregulation correlates with increased intestinal permeability, sustained activation of inflammation, epithelial-to-mesenchymal transition (EMT), and tumor progression in IBD as well as consequent colitis-associated colorectal cancer (CAC), possibly shedding new light on further etiologic research and clinical treatments.
Histopathologic determinants of regional lymph node metastasis in early colorectal cancer.
Ishikawa Yukio,Akishima-Fukasawa Yuri,Ito Kinji,Akasaka Yoshikiyo,Yokoo Tomoko,Ishii Toshiharu,
BACKGROUND:Early colorectal cancer (ECC) is curable by endoscopic local resection; however, 10% of patients with ECC exhibit lymph node (LN) metastasis. In the current study, accurate predictors for LN metastasis in patients with ECC were examined by using immunohistochemistry with the lymphatic endothelial hyaluronan receptor 1 (LYVE-1) antibody to discriminate between lymphatics and blood vessels. METHODS:Colorectal tissue specimens obtained from 71 patients with ECC, including 28 patients with regional LN metastasis, were immunostained with antibodies against LYVE-1, beta-catenin, claudin-3, claudin-4, and cytokeratin. The significance of the histopathologic variables for LN metastasis in ECC was investigated on the basis of specific histopathologic parameters. RESULTS:Lymphatic invasion confirmed by LYVE-1 immunohistochemistry was observed mainly in the submucosal area around the primary tumor and rarely was observed in the tumor. Expression patterns of beta-catenin, claudin-3, and claudin-4 in cancer cells at the invasive front were irrelevant to LN status. Tumor size, depth of invasion, histologic tumor type, budding formation, and lymphatic invasion were statistically significant to LN status in univariate analysis; however, only 2 factors--lymphatic invasion and budding formation at the invasive front-were independent predictors of LN metastasis in ECC. CONCLUSIONS:LYVE-1 immunohistochemistry appeared to be a useful method for detecting lymphatics invaded by cancer cells, and detailed examination of the submucosa around the tumor may be important for predicting LN metastasis. When lymphatic invasion and budding formation are observed histopathologically in patients with ECC, additional therapy, such as adjuvant chemotherapy or a curative resection of the regional LN, may be required.
Claudin-1 protein is a major factor involved in the tumorigenesis of colorectal cancer.
Huo Qun,Kinugasa Tetsushi,Wang Lin,Huang Jian,Zhao Jun,Shibaguchi Hirotomo,Kuroki Motomu,Tanaka Toshihiro,Yamashita Yuichi,Nabeshima Kazuki,Iwasaki Hiroshi,Kuroki Masahide
BACKGROUND:The molecular and morphological alterations of the tight junctions in colorectal cancer (CRC) are still poorly understood. The possible involvement of claudin-1 (CL-1), one of the major tight junctional proteins (TJPs), was investigated in the tumorigenesis of CRC. PATIENTS AND METHODS:Adenocarcinoma tissue and paired normal mucosa specimens were resected from surgical specimens of CRC patients and analyzed to determine whether the expression of CL-1 correlated with the clinicopathological factors and to determine the role of CL-1 in the alteration of tight junctions during tumorigenesis. RESULTS:The expression of CL-1 at the mRNA and protein levels was analyzed in 41 cases and was found to increase in the CRC tissue in comparison to that in the normal tissue specimens. The mRNA levels of CL-1 were correlated with tumor depth, but not with the preoperative carcinoembryonic antigen (CEA) serum level. When T84 cells, a human colon cancer cell line, were transfected with the CL-1 gene, the CL-1 overexpressing cells grew as aggregates in contrast to the monolayer formation of the parental cells. In addition, trypsin-treated CL-1 overexpressing cells aggregated more easily than did the parental cells. CONCLUSION:CL-1 plays a pivotal role in cell morphology and behavior in the colonic epithelium. CL-1 protein may therefore be one of the major factors involved in the tumorigenesis of CRC.
Prostaglandin E2-EP1 and EP2 receptor signaling promotes apical junctional complex disassembly of Caco-2 human colorectal cancer cells.
Tanaka Marcelo N,Diaz Bruno L,de Souza Wanderley,Morgado-Diaz Jose A
BMC cell biology
BACKGROUND:The apical junctional complex (AJC) is a dynamic structure responsible to maintain epithelial cell-cell adhesions and it plays important functions such as, polarity, mechanical integrity, and cell signaling. Alteration of this complex during pathological events leads to an impaired epithelial barrier by perturbation of the cell-cell adhesion system. Although clinical and experimental data indicate that prostaglandin E(2) (PGE2) plays a critical function in promoting cell motility and cancer progression, little is known concerning its role in AJC disassembly, an event that takes place at the beginning of colorectal tumorigenesis. Using Caco-2 cells, a cell line derived from human colorectal cancer, we investigated the effects of prostaglandin E(2) (PGE(2)) treatment on AJC assembly and function. RESULTS:Exposition of Caco-2 cells to PGE(2) promoted differential alteration of AJC protein distribution, as evidenced by immunofluorescence and immunoblotting analysis and impairs the barrier function, as seen by a decrease in the transepithelial electric resistance and an increase in the permeability to ruthenium red marker. We demonstrated the involvement of EP1 and EP2 prostaglandin E(2) receptor subtypes in the modulation of the AJC disassembly caused by prostanoid. Furthermore, pharmacological inhibition of protein kinase-C, but not PKA and p38MAPK significantly prevented the PGE(2) effects on the AJC disassembly. CONCLUSION:Our findings strongly suggest a central role of Prostaglandin E2-EP1 and EP2 receptor signaling to mediate AJC disassembly through a mechanism that involves PKC and claudin-1 as important target for the TJ-related effects in human colorectal cancer cells (Caco-2).
Increased claudin-1 protein expression contributes to tumorigenesis in ulcerative colitis-associated colorectal cancer.
Kinugasa Tetsushi,Akagi Yoshito,Yoshida Takefumi,Ryu Yasuhiko,Shiratuchi Ichitarou,Ishibashi Nobuya,Shirouzu Kazuo
BACKGROUND:The molecular and morphological alterations of the tight junctions in ulcerative colitis (UC)-associated colorectal cancer are still poorly understood. The possible involvement of claudin-1 (CL-1), one of the major tight junctional proteins, was investigated in the tumorigenesis of UC-associated CRC. PATIENTS AND METHODS:A total of 39 patients with UC underwent surgical treatment from January 2001 until October 2009 at Kurume University Hospital in Fukuoka. CRC tissue specimens were analyzed to determine whether the expression of CL-1 correlates with clinicopathological factors and to determine the role of CL-1 and β-catenin in the alteration of tight junctions during tumorigenesis. RESULTS:The operations were 30 of elective surgery and 9 of emergency surgery. Colectomy was performed in five patients (12.8%) because of UC-associated CRC, and in another patient (2.6 %) because of high-grade dysplasia. The immunostaining pattern of the high-grade dysplasia and UC-associated CRC for CL-1 showed much stronger and more diffuse staining in comparison to the normal or UC colonic mucosa. The expression of β-catenin was also positive or up-regulated in all of the UC-associated CRC and high-grade dysplasia tissue specimens. CONCLUSION:These observations suggested that CL-1 plays a pivotal role in the regulation of cellular morphology and behavior in UC. We speculate that increased CL-1 expression may be involved in the early stages of transformation in UC-associated neoplasia. CL-1 protein may therefore be a good candidate for surveillance of patients with UC.
Claudin-1 promotes TNF-α-induced epithelial-mesenchymal transition and migration in colorectal adenocarcinoma cells.
Bhat Ajaz A,Ahmad Rizwan,Uppada SrijayaPrakash B,Singh Amar B,Dhawan Punita
Experimental cell research
Epithelial-mesenchymal transition (EMT) is an important mechanism in cancer progression and malignancy including colorectal cancer (CRC). Importantly, inflammatory mediators are critical constituents of the local tumor environment and an intimate link between CRC progression and inflammation is now validated. We and others have reported key role of the deregulated claudin-1 expression in colon carcinogenesis including colitis-associated colon cancer (CAC). However, the causal association between claudin-1 expression and inflammation-induced colon cancer progression remains unclear. Here we demonstrate, TNF-α, a pro-inflammatory cytokine, regulates claudin-1 to modulate epithelial to mesenchymal transition (EMT) and migration in colon adenocarcinoma cells. Importantly, colon cancer cells cultured in the presence of TNF-α (10ng/ml), demonstrated a sharp decrease in E-cadherin expression and an increase in vimentin expression (versus control cells). Interestingly, TNF-α treatment also upregulated (and delocalized) claudin-1 expression in a time-dependent manner accompanied by increase in proliferation and wound healing. Furthermore, similar to our previous observation that claudin-1 overexpression in CRC cells induces ERK1/2 and Src- activation, signaling associated with colon cancer cell survival and transformation, TNF-α-treatment induced upregulation of phospho-ERK1/2 and -Src expression. The shRNA-mediated inhibition of claudin-1 expression largely abrogated the TNF-α-induced changes in EMT, proliferation, migration, p-Erk and p-Src expression. Taken together, our data demonstrate TNF-α mediated regulation of claudin-1 and tumorigenic abilities of colon cancer cells and highlights a key role of deregulated claudin-1 expression in inflammation-induced colorectal cancer growth and progression, through the regulation of the ERK and Src-signaling.
Defective claudin-7 regulation by Tcf-4 and Sox-9 disrupts the polarity and increases the tumorigenicity of colorectal cancer cells.
Darido Charbel,Buchert Michael,Pannequin Julie,Bastide Pauline,Zalzali Hassan,Mantamadiotis Theo,Bourgaux Jean-François,Garambois Véronique,Jay Philippe,Blache Philippe,Joubert Dominique,Hollande Frédéric
Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.
An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.
Han Chia-Li,Chen Jinn-Shiun,Chan Err-Cheng,Wu Chien-Peng,Yu Kun-Hsing,Chen Kuei-Tien,Tsou Chih-Chiang,Tsai Chia-Feng,Chien Chih-Wei,Kuo Yung-Bin,Lin Pei-Yi,Yu Jau-Song,Hsueh Chuen,Chen Min-Chi,Chan Chung-Chuan,Chang Yu-Sun,Chen Yu-Ju
Molecular & cellular proteomics : MCP
We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.
Increased claudin-1 protein expression in hepatic metastatic lesions of colorectal cancer.
Kinugasa Tetsushi,Akagi Yoshito,Ochi Takafumi,Tanaka Natsuki,Kawahara Akihiko,Ishibashi Yoshinobu,Gotanda Yukito,Yamaguchi Keizo,Shiratuchi Ichitarou,Oka Yousuke,Kage Masayoshi,Shirouzu Kazuo
BACKGROUND:The molecular and morphological alterations of the tight junctions in hepatic metastatic lesions are poorly understood. The possible involvement of claudin-1 (CL-1), which is one of the major tight junctional proteins, was investigated in the tumorigenesis of hepatic metastasis in patients with colorectal cancer (CRC). PATIENTS AND METHODS:A total of 14 patients with hepatic metastasis of CRC who underwent surgical treatment from January 2007 until December 2010 at the Kurume University Hospital in Fukuoka, were examined. CRC tissue specimens were analyzed to determine whether the levels of CL-1 correlated with clinicopathological factors and to determine the roles of CL-1, β-catenin, and E-cadherin in the alterations of the tight junctions during tumorigenesis. RESULTS:In seven cases, the tumors were located in the colon, while the other seven tumors were found in the rectum. There were eight cases of synchronous liver metastasis, while there were six cases of metachronous liver metastasis. The levels of the CL-1 protein were up-regulated in CRC and in hepatic metastatic lesions. The levels of β-catenin were positive or up-regulated in the primary CRC lesions and in hepatic metastatic lesions. Despite the finding that the levels of E-cadherin were decreased in CRC, they were clearly up-regulated in hepatic metastatic lesions in this study. CONCLUSION:This study demonstrated that CL-1 levels were up-regulated in liver metastatic lesions from primary CRC lesions. Moreover, the levels of E-cadherin were increased in liver metastatic lesions, which may point to the existence of interactions between CL-1 and E-cadherin in hepatic metastatic lesions. These observations suggest that CL-1 plays a pivotal role in the regulation of cellular morphology and in the behavior of metastatic processes in CRC.
Expression of claudin-1, -3, -4, -5 and -7 proteins in low grade colorectal carcinoma of canines.
Jakab Cs,Rusvai M,Gálfi P,Szabó Z,Szabára A,Kulka J
Histology and histopathology
UNLABELLED:The aim of the present study was to characterise the expression pattern of claudin-1, -3, -4, -5 and -7 tight junction proteins in canine normal colorectum and in the low-grade, tubulopapillary colorectal carcinoma in canines. METHODS AND RESULTS:The biopsy samples included 10 canine normal colorectal tissues and 20 canine low grade colorectal carcinomas (CLGCCs). The canine normal colorectal mucosa was negative for claudin-1. Claudin-1 was detected as a non-diffuse intense membrane labelling of neoplastic epithelial cells in low grade colorectal cancer in canines. Fifty five per cent of all tumours showed a weak cytoplasmic pattern of staining for claudin-1 protein. The normal colorectal mucosa showed diffuse punctate positivity for claudin-3. Claudin-3 was detected as an intense lateral membrane labelling of tumour cells in CLGCCs. Claudin-4 expression in surface and crypt epithelial cells of the intact colorectal mucosa in canines was punctate. Claudin-4 molecule was detected as a lateral membrane labelling of neoplastic cells in CLGCCs. The epithelium of the CLGCCs and the low grade colorectal carcinoma were negative for claudin-5. The surface and crypt epithelial cells of the canine normal colorectal mucosa showed a diffuse lateral membranous pattern of staining for claudin-7. Claudin-7 molecule was detected as an intense membrane labelling of neoplastic cells in CLGCCs. Seventy per cent of all tumours showed weak cytoplasmic positivity for claudin-7. CONCLUSION:Consequently, we hypothesize that claudin-1 plays a role in the progression of CLGCCs. Further functional studies are needed to clarify the biological role of the mislocalization of the claudin-1 molecule from cell membrane to the cytoplasm in CLGCCs. Lower claudin-4 expression suggests that reduced expression of claudin-4 molecule may lead to cellular disorientation, detachment and invasion of CLGCCs. Further functional studies are needed to clarify the biological role of overexpression and mislocalisation of claudin-7 in CLGCCs.
Differential expression of genes encoding tight junction proteins in colorectal cancer: frequent dysregulation of claudin-1, -8 and -12.
Gröne J,Weber B,Staub E,Heinze M,Klaman I,Pilarsky C,Hermann K,Castanos-Velez E,Röpcke S,Mann B,Rosenthal A,Buhr H J
International journal of colorectal disease
BACKGROUND AND AIMS:As integral membrane proteins, claudins form tight junctions together with occludin. Several claudins were shown to be up-regulated in various cancer types. We performed an expression analysis of genes encoding tight junction proteins to display differential gene expression on RNA and protein level and to identify and validate potential targets for colorectal cancer (CRC) therapy. PATIENTS AND METHODS:Amplified and biotinylated cRNA from 30 microdissected CRC specimen and corresponding normal tissues was hybridized to Affymetrix U133set GeneChips. Quantification of differential protein expression of claudin-1, -8 and -12 between normal and corresponding tumour tissues was performed by Western blot analyses. Paraffin-embedded CRC tissue samples, colon cancer cell lines and normal tissue microarray were analysed for protein expression of claudin-1 by immunohistochemistry (IHC). RESULTS:Claudin-1 (CLDN1) and -12 (CLDN12) are frequently overexpressed in CRC, whereas claudin-8 (CLDN8) shows down-regulation in tumour tissue on RNA level. Quantification of proteins confirmed the overexpression of claudin-1 in tumour tissues, whereas changes of claudin-8 and -12 were not significantly detectable on protein level. IHC confirmed the markedly elevated expression level of claudin-1 in the majority of CRC, showing membranous and intracellular vesicular staining. CONCLUSIONS:Differential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.
gene knockout causes destruction of intestinal structure and animal death in mice.
Xu Chang,Wang Kun,Ding Yu-Han,Li Wen-Jing,Ding Lei
World journal of gastroenterology
BACKGROUND:Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of knockout mice using the Cre/LoxP system. AIM:To determine the function of the tumor suppressor gene by generating three lines of gene knockout mice. METHODS:We crossed -floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional knockout mice, conditional (intestinal specific) knockout mice, and inducible conditional knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional knockout mice can induce the knockout of . PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of . The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin (HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers. RESULTS:We generated -floxed mice and three lines of gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional knockout mice was increased compared with control mice (small intestine: 54.1 ± 2.96 38.4 ± 1.63; large intestine: 44.7 ± 1.93 27.4 ± 0.60; < 0.001). CONCLUSION:The knockout of causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice. may act as a tumor suppressor gene in the development of colorectal cancer.
Expression of CLDN1 in colorectal cancer: a novel marker for prognosis.
Nakagawa Sumiko,Miyoshi Norikatsu,Ishii Hideshi,Mimori Koshi,Tanaka Fumiaki,Sekimoto Mitsugu,Doki Yuichiro,Mori Masaki
International journal of oncology
Claudin1 (CLDN1) plays an important role not only in the intercellular barrier function of tight junctions (TJs) but also in migration and invasiveness of cancer cells. Previous reports show that CLDN1 overexpression is significantly related to the malignant behavior in several cancer types whereas its significance in colorectal cancer (CRC) is not fully understood. The present study comprised 119 patients who underwent surgery for CRC, as well as 3 cell lines derived from human CRC. The correlation of gene expression with clinical parameters in patients was assessed by knockdown experiments using 3 cell lines. Patients with high CLDN1 expression were statistically shown to have a relatively better prognosis, and those with low CLDN1 expression showed poorer overall survival and disease-free survival than those with high expression. The assessment of CLDN1 knockdown in the 3 cell lines demonstrated that the siRNA inhibition resulted in a statistically significant increase in cell invasiveness. In conclusion, the present data strongly suggest that CLDN1 expression is a prognostic factor in CRC patients.
SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.
Wang Yaxi,Sun Tingyi,Sun Haimei,Yang Shu,Li Dandan,Zhou Deshan
International journal of molecular sciences
Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.
Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer.
Kinugasa Tetsushi,Huo Qun,Higashi Daijiro,Shibaguchi Hirotomo,Kuroki Motomu,Tanaka Toshihiro,Futami Kitarou,Yamashita Yuichi,Hachimine Ken,Maekawa Shinichi,Nabeshima Kazuki,Iwasaki Hiroshi,Kuroki Masahide
BACKGROUND:To understand the molecular and morphological alterations in the tight junction in colorectal cancer (CRC) tissues, the expression of eight tight junction proteins in normal and cancer colorectal tissues were compared. PATIENTS AND METHODS:Adenocarcinoma tissues and paired normal mucosa were resected from surgical specimens of CRC patients. The expression of occludin, ZO-1, ZO-2, and claudin-1 -5 was analyzed at the mRNA level by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry. RESULTS:The expression of claudin-1 and claudin-2 in cancer tissues was upregulated 40- and 49.2-fold, respectively, at the mRNA level, as compared with that in normal tissues. The up-regulation of these two claudins was also observed at the protein level and it appeared to depend on the depth of tumor invasion. CONCLUSION:Claudin-1 and claudin-2 were found to be overexpressed in CRC tissues. They may be useful as tumor markers and targets for the treatment of colorectal cancer.
The level of claudin-7 is reduced as an early event in colorectal carcinogenesis.
Bornholdt Jette,Friis Stine,Godiksen Sine,Poulsen Steen S,Santoni-Rugiu Eric,Bisgaard Hanne C,Lothe Inger M B,Ikdahl Tone,Tveit Kjell M,Johnson Egil,Kure Elin H,Vogel Lotte K
BACKGROUND:Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both in vivo and in vitro. We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression. We have previously shown that the matriptase expression level decreases during colorectal carcinogenesis. In the present study we investigated whether claudin-7 expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue. METHODS:The mRNA level of claudin-7 (CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings. RESULTS:A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the claudin-7 mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings. CONCLUSIONS:Our results show that the claudin-7 mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.
Upregulation of microRNA-155 promotes the migration and invasion of colorectal cancer cells through the regulation of claudin-1 expression.
Zhang Guang-Jun,Xiao Hua-Xu,Tian Hong-Peng,Liu Zuo-Liang,Xia Shu-Sen,Zhou Tong
International journal of molecular medicine
Human microRNA-155 (miR-155) has been demonstrated to regulate a variety of cellular functions, including epithelial-to-mesenchymal transition (EMT) by targeting multiple messenger RNAs (mRNAs). However, its role in colorectal cancer (CRC) remains unelucidated. Therefore, the aim of the present study was to investigate the effects of miR-155 on CRC cells. The expression level of miR-155 was quantified by quantitative real-time reverse transcriptase-PCR (qRT-PCR) in primary CRC tissues and normal adjacent mucosa. MTT, migration and invasion assays were used to examine the proliferation, migration and invasion of SW480 cells transfected with miR‑155. The expression of miR-155 was significantly upregulated in the CRC tissues and the high expression of miR-155 correlated with an advanced clinical stage, lymph node and distant metastases. The ectopic expression of miR-155 enhanced the migration and invasive ability of the SW480 cells and altered their morphological appearance; however, cell proliferation was not affected. E-cadherin expression levels decreased, while ZEB1 expression levels increased in the SW480 cells overexpressing miR-155. Furthermore, the overexpression of miR-155 upregulated claudin-1 expression. Thus, our data suggest that miR-155 plays an important role in promoting CRC cell migration and invasion, at least in part through the regulation of claudin-1 expression and controlling metastasis in CRC.
A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression.
Kuhn Sebastian,Koch Moritz,Nübel Tobias,Ladwein Markus,Antolovic Dalibor,Klingbeil Pamela,Hildebrand Dagmar,Moldenhauer Gerhard,Langbein Lutz,Franke Werner W,Weitz Jürgen,Zöller Margot
Molecular cancer research : MCR
High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7-associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation.
Epithelial tight junctional changes in colorectal cancer tissues.
Wang Xuexuan,Tully Owen,Ngo Benjamin,Zitin Marc,Mullin James M
Colorectal cancer (CRC) is one of the most common cancers in the western world. Early screening and detection could be highly preventative and therefore reduce mortality. Tight junctions (TJ) are well known for their function in controlling paracellular traffic of ions and molecules. It has become increasingly evident that TJs play a crucial role in maintaining cell-cell integrity, and the loss of cell junctional sealing could involve itself in the processes of carcinoma and cancer metastasis. If correlations between altered TJ proteins and CRC presence or invasiveness could be established, they may serve as important markers and guidelines for prophylactic and prognostic purposes, along with other screening methods. This review will present recent data from clinical and animal studies showing how altered TJ protein expression is a feature of certain CRCs. The up-regulation of claudin-1 in many CRCs is especially noteworthy. The focus of this article is simply on the association - however imperfect - between CRC and the major TJ transmembrane barrier proteins, namely claudins and occludin. Any causal relationship between TJ protein change and neoplasia remains conclusively unproven at present.
Claudin-3 overexpression increases the malignant potential of colorectal cancer cells: roles of ERK1/2 and PI3K-Akt as modulators of EGFR signaling.
de Souza Waldemir F,Fortunato-Miranda Natalia,Robbs Bruno K,de Araujo Wallace M,de-Freitas-Junior Julio C,Bastos Lilian G,Viola João P B,Morgado-Díaz José A
The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways.
Immunohistochemical analysis of colorectal cancer with gastric phenotype: claudin-18 is associated with poor prognosis.
Matsuda Miho,Sentani Kazuhiro,Noguchi Tsuyoshi,Hinoi Takao,Okajima Masazumi,Matsusaki Keisuke,Sakamoto Naoya,Anami Katsuhiro,Naito Yutaka,Oue Naohide,Yasui Wataru
Claudin-18 plays a key role in constructing tight junctions, and altered claudin-18 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin-18 in colorectal cancer (CRC). The aim of this study is to investigate the significance of claudin-18 expression in CRC and its association with clinicopathological factors. We performed clinicopathological analysis of claudin-18 expression in a total of 569 CRCs by immunohistochemistry. Moreover, we investigated the association between claudin-18 and various markers including gastric/intestinal phenotype (MUC5AC, MUC6, MUC2 and CD10), CDX2, claudin-3, claudin-4, p53 and Ki-67. Claudin-18 expression was detected in 21 of the 569 CRCs (4%) and was seen exclusively on the cell membrane. Positive expression of claudin-18 showed a significant correlation with positive expression of MUC5AC (P < 0.0001) and negative expression of CDX2 (P= 0.0013). The prognosis of patients with positive claudin-18 expression was significantly poorer than in negative cases (P= 0.0106). Multivariate analysis revealed that T grade, M grade and claudin-18 expression were independent predictors of survival in patients with CRC. We revealed that claudin-18 expression correlates with poor survival in patients with CRC and is associated with the gastric phenotype.
Claudins upregulation in human colorectal cancer.
de Oliveira Silvia Souza,de Oliveira Ivanir Martins,De Souza Wanderley,Morgado-Díaz José Andrés
In colorectal cancer tight junction molecular and morphological alterations are poorly understood. In this study, adenocarcinoma tissues and their paired normal mucosa (n = 12) were analyzed for tight junction alterations molecular. The expression of claudin-1, -3 and -4 was upregulated 5.7-, 1.5- and 2.4-fold, respectively, in colorectal tumor tissues in comparison to the normal ones. Although tight junction remains in the cancerous epithelium, its barrier function was altered. Despite claudins overexpression, paracellular permeability to ruthenium red was increased and a significant disorganization of tight junction strands was observed in freeze fracture replicas. Whereas the functional significance of claudin overexpression in colorectal cancer is unclear, these proteins can become potential markers and targets in colorectal cancer.
Loss of tight junction proteins (Claudin 1, 4, and 7) correlates with aggressive behavior in colorectal carcinoma.
Süren Dinç,Yıldırım Mustafa,Kaya Vildan,Alikanoğlu Arsenal Sezgin,Bülbüller Nurullah,Yıldız Mustafa,Sezer Cem
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND:Tight junction proteins in the cell organize paracellular permeability and they play a critical role in apical cell-to-cell adhesion and epithelial polarity. Claudins are major integral membrane proteins of tight junctions, especially Claudin 1, 4, and 7, which are known as the impermeability Claudins. In this study, we investigated the importance of loss of Claudin 1, 4, and 7 expression, and their relation to tumor progression in colorectal cancer patients. MATERIAL/METHODS:Loss of Claudin 1, 4, and 7 expression was examined by immunohistochemical method in 70 patients diagnosed with colorectal cancer. Cases with loss of Claudin expression in <1/3 of tumor cells were classified as mild loss, whereas cases with loss of Claudin expression ³1/3 of tumor cells were classified as moderate-to-marked loss in order to evaluate the relation between loss of Claudin 1, 4, and 7 expression and clinicopathologic data. RESULTS:The severe suppression of Claudin 1, 4, and 7 expression was found to be significantly related to the depth of tumor invasion, positive regional lymph nodes, histological grade, lymphovascular invasion, perineural invasion, and lymphocytic response. Additionally, severity of loss in Claudin 4 expression was found to have a relation with distant metastasis. CONCLUSIONS:Claudin 1, 4, and 7 are important building blocks of paracellular adhesion molecules. Their decreased expression in colorectal cancer seems to have critical effects on cell proliferation, motility, invasion, and immune response against the tumor.
Reduced expression of the claudin-7 gene correlates with venous invasion and liver metastasis in colorectal cancer.
Oshima Takashi,Kunisaki Chikara,Yoshihara Kazue,Yamada Roppei,Yamamoto Naoto,Sato Tsutomu,Makino Hirochika,Yamagishi Shigeru,Nagano Yasuhiko,Fujii Shoich,Shiozawa Manabu,Akaike Makoto,Wada Nobuyuki,Rino Yasushi,Masuda Munetaka,Tanaka Katsuaki,Imada Toshio
Claudins, members of a large family of adherent junction proteins, regulate the integrity and function of tight junctions and influence tumorigenesis. Studies have suggested that altered levels of different claudins are related to carcinoma-cell invasion and disease progression. This study examined the relationship between the relative expression of claudin genes and clinicopathological factors, especially invasion and metastasis, in patients with colorectal cancer. We studied surgical specimens of cancer tissue and adjacent normal mucosa from 205 patients with untreated colorectal carcinoma. The relative expression levels of claudin-1, -3, -4 and -7 mRNA in cancer and in normal adjacent mucosa were measured by quantitative real-time, reverse-transcription polymerase chain reaction. The relative expression levels of the claudin-1, -3 and -4 genes were higher in cancer than in normal adjacent mucosa, whereas the relative expression of the claudin-7 gene was similar. An analysis of the relationship between the clinicopathological features and gene expression showed that reduced expression of claudin-7 correlated with venous invasion and liver metastasis. There was also a correlation between claudin-3 and -4 gene expression. Our results suggested that a reduced expression of the claudin-7 gene might lead to venous invasion and liver metastasis in colorectal cancer. Reduced expression of the claudin-7 gene may thus be a useful predictor of liver metastasis in patients with colorectal cancer.
Hypoxia-induced vasculogenic mimicry formation in human colorectal cancer cells: Involvement of HIF-1a, Claudin-4, and E-cadherin and Vimentin.
Li Wen,Zong ShaoQi,Shi Qi,Li HongJia,Xu Jian,Hou Fenggang
Vasculogenic mimicry (VM) plays an important role in colorectal cancer (CRC) metastasis, and both hypoxia and the epithelial-mesenchymal transition (EMT) are necessary for VM. In this study, HIF-1α expression was upregulated in the VM-positive CRC cell line HCT-116 and thereby affected the expression of the EMT-related markers Claudin-4, E-cadherin (E-cd) and Vimentin(VIM). SB431542 and U0126EtOH, which can inhibit of EMT were used to treat HCT-116 and HCT-8 in these experiments. Both of the inhibitors had significant effect on EMT markers and the formations of VM in CRC cells. In addition, knockdown of HIF-1α in the HCT-116 cells inhibited their capacity for VM. Our study reveals a regulatory role for HIF-1α in VM and suggests that targeting either HIF-1α or EMT may be a valuable strategy for the elimination of CRC metastasis.
Prognostic and clinical significance of claudin-1 in colorectal cancer: A systemic review and meta-analysis.
Jiang Lu,Yang Long,Huang He,Liu Bai-Ying,Zu Guo
International journal of surgery (London, England)
BACKGROUND:The current reports on the association of claudin-1 expression with colorectal cancer (CRC) result were controversial. Thus, we conducted a meta-analysis to assess the correlation between claudin-1 expression and the clinical parameters and assess the prognostic value of claudin-1 in CRC. METHODS:Systematic searches on PubMed, Embase, Elsevier, CNKI (China National Knowledge Infrastructure), Wanfang data and Cochrane Library prior to August 2016 were performed. The pooled odds ratio (OR) with its 95% confidence interval (95 %CI) was used to assess association between claudin-1 expression and clinical parameters of CRC patients, and to assess association between claudin-1 expression and the prognostic value of CRC patients. RESULTS:Eight studies with a total of 1146 CRC patients were included. Overall, the pooled results showed that low expression of claudin-1 was associated with TNM III-IV stage of CRC patients (OR: 1.714, 95%CI: 1.215-2.418, P = 0.002). Low expression of claudin-1 was also associated with a poor survival in CRC patients (one year survival rate: OR: 2.112, 95%CI: 1.028-4.339, P = 0.042; three years survival rate: OR: 1.501, 95%CI: 1.030-2.186, P = 0.035; five years survival rate: OR: 1.794, 95%CI: 1.139-2.439, P = 0.000). Whereas, low expression of claudin-1 is not associated with gender (OR: 1.259, 95%CI: 0.957-1.657, P = 0.100), tumors' differentiation (OR: 1.317, 95%CI: 0.916-1.892, P = 0.137), depth of invasion (OR: 1.016, 95 %CI: 0.701-1.472, P = 0.935) and lymph node metastasis group (OR: 1.286, 95% CI: 0.982-1.684, P = 0.06) of CRC. CONCLUSIONS:Low expression of claudin-1 is associated with TNM III-IV stage and poor prognosis of CRC patients. Low expression of claudin-1 is not associated with gender, tumors' differentiation depth of invasion and lymph node involvement of CRC patients.
Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis.
Georges Rania,Bergmann Frank,Hamdi Hadjar,Zepp Michael,Eyol Ergül,Hielscher Thomas,Berger Martin R,Adwan Hassan
Journal of cellular and molecular medicine
Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I-III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.
Dysregulation of Claudin family genes in colorectal cancer in a Chinese population.
Tang Weijun,Dou Tonghai,Zhong Ming,Wu Zhiyong
BioFactors (Oxford, England)
Claudins play an important role in tumor metastasis and in invasiveness of colorectal cancer (CRC). We have evaluated the relationship between CRC and expression of the claudin genes in Chinese patients with CRC. We measured CLDN1 and CLDN7 mRNA using quantitative PCR, and protein levels with immunohistochemistry in cancer tissues and adjacent normal tissue. Cancer tissues had significantly higher levels of CLDN1, and significantly lower levels of CLDN3, CLDN4, and CLDN7 than did normal tissue. CLDN3, CLDN4, and CLDN7 expression levels were higher in CRC of the protruded type than in CRC of the infiltrative type. Expression of CLDN7 correlated with lymph node metastasis. Stage N0 cancer tissues had higher levels of CLDN7 than did stages N1 and N2, suggesting that CLDN7 expression was closely related to the extent of lymph node metastasis. CLDN1 protein was upregulated, but CLDN7 protein was downregulated in cancer tissues when compared with expression in adjacent normal tissues. In conclusion, CLDN3, CLDN4, and CLDN7 were significantly downregulated, whereas CLDN1 was significantly upregulated in CRC. The altered expression of claudin genes may play a role in the initiation and development of CRC.
Low expression of claudin-1 and presence of poorly-differentiated tumor clusters correlate with poor prognosis in colorectal cancer.
Shibutani Masatsune,Noda Eiji,Maeda Kiyoshi,Nagahara Hisashi,Ohtani Hiroshi,Hirakawa Kosei
BACKGROUND:Claudins are integral membrane proteins that constitute tight junctions. The aim of this study was to determine the clinical significance of claudin-1 in terms of the cell-cell adhesion, considering the poorly-differentiated tumor component. PATIENTS AND METHODS:A total of 344 cases of stage II and III colorectal cancer were enrolled. We evaluated the expression of claudin-1 by immunohistochemistry and the extent of the poorly differentiated component. RESULTS:Low expression of claudin-1 was associated with lymphatic involvement, histological differentiation, extent of poorly-differentiated component, reduced disease-free and overall survival. Multivariate analysis indicated that lymph node metastasis, venous involvement and low claudin-1 expression were independent predictors of recurrence, and that lymph node metastasis, venous involvement and low claudin-1 expression were associated with poor survival. CONCLUSION:Our study not only showed the significance of low expression of claudin-1 as a predictor of poor prognosis of colorectal cancer but also supported the concept of grading of the poorly-differentiated component.
Loss of claudin-1 expression induces epithelial-mesenchymal transition through nuclear factor-κB activation in colorectal cancer.
Kim Nae Yu,Pyo Jung-Soo,Kang Dong-Wook,Yoo Seung-Min
Pathology, research and practice
OBJECTIVE:The aim of this study was to elucidate the clinicopathological significance and prognostic role of loss of claudin-1 in colorectal cancer (CRC). METHODS:The correlations between claudin-1 expression and clinicopathological characteristics, including survival rates, were assessed using immunohistochemistry on 260 archival, paraffin-embedded CRC tissues. In addition, the correlations between cludin-1 and nuclear factor-kappa B (NF-κB), epithelial-mesenchymal transition markers and tumor-infiltrating lymphocytes were investigated. RESULTS:Claudin-1 expression was markedly lost in 42.7% of the 260 CRCs analyzed. Loss of claudin-1 expression significantly correlated with larger tumor size, vascular invasion, higher pT stage, and high metastatic lymph node ratio. In addition, loss of claudin-1 expression significantly correlated with NF-κB activation (P < 0.001), high SNAI (P < 0.001), and low E-cadherin (P < 0.001) expressions. Patients with high immunoscores showed significantly lower rates of claudin-1 expression loss (P = 0.020). In detail, loss of claudin-1 expression were frequently found in CRCs low CD3- and CD8-positive lymphocytes. There were significant correlations between claudin-1 expression loss and poor overall and recurrence-free survivals (P < 0.001 and P < 0.001, respectively). CONCLUSION:Taken together, our results suggest that the loss of claudin-1 expression significantly correlates with aggressive tumor behaviors, high SNAI expression, lower immunoscore, and poor prognoses.
Claudin-7 promotes the epithelial-mesenchymal transition in human colorectal cancer.
Philip Rahel,Heiler Sarah,Mu Wei,Büchler Markus W,Zöller Margot,Thuma Florian
In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tumor-promoting EpCAM activities are modulated by the association with claudin-7. To support this hypothesis, claudin-7 was knocked-down (kd) in HT29 and SW948 cells. HT29-cld7kd and SW948-cld7kd cells display decreased anchorage-independent growth and the capacity for holoclone-, respectively, sphere-formation is reduced. Tumor growth is delayed and cld7kd cells poorly metastasize. In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells. This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM. The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells. But, uptake of HT29wt and SW948wt exosomes by the claudin-7kd lines sufficed for transcription factor upregulation and for restoring motility. Thus, claudin-7 contributes to motility and invasion and is required for recruiting EpCAM towards TACE/presenilin2. EpIC generation further supports motility by promoting a shift towards EMT. Notably, EMT features of cld7-competent metastatic CoCa cells can be transferred via exosomes to poorly metastatic cells.
Antibody targeting of claudin-1 as a potential colorectal cancer therapy.
Cherradi S,Ayrolles-Torro A,Vezzo-Vié N,Gueguinou N,Denis V,Combes E,Boissière F,Busson M,Canterel-Thouennon L,Mollevi C,Pugnière M,Bibeau F,Ychou M,Martineau P,Gongora C,Del Rio M
Journal of experimental & clinical cancer research : CR
BACKGROUND:Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). METHODS:Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. RESULTS:Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. CONCLUSION:Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target.
Tight Junction Protein Claudin-2 Promotes Self-Renewal of Human Colorectal Cancer Stem-like Cells.
Paquet-Fifield Sophie,Koh Shir Lin,Cheng Lesley,Beyit Laura M,Shembrey Carolyn,Mølck Christina,Behrenbruch Corina,Papin Marina,Gironella Meritxell,Guelfi Sophie,Nasr Ramona,Grillet Fanny,Prudhomme Michel,Bourgaux Jean-Francois,Castells Antoni,Pascussi Jean-Marc,Heriot Alexander G,Puisieux Alain,Davis Melissa J,Pannequin Julie,Hill Andrew F,Sloan Erica K,Hollande Frédéric
Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDH CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDH phenotype. Next-generation sequencing in ALDH cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer. Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. .
[Еxpression of claudin-1, 3, and 4 in colorectal cancer and polyps].
Nefedova N А,Kharlova О А,Malkov P G
UNLABELLED:Claudins are a family of transmembrane tight junctions proteins. It is proven that claudins undergo structural and functional alteration in malignant cells. However, very few researches are pursued on this topic, the data provided by different researchers are controversial. The aim of this study was to evaluate expression of tight junction proteins in cancer and benign polyps of the colon and rectum. MATERIAL AND METHODS:Specimens of 32 colorectal adenocarcinomas and biopsy specimens of 86 polypoid lesions of the colon and rectum were selected from diagnostic material. Polyps were divided into 6 groups following the 2010 WHO classification of premalignant lesions of the colon and rectum. Immunohistochemical labeling with claudin-1, claudin-3 and claudin-4 antibodies was performed in all cases. We used G. Sheehan et al. (2007) method to evaluate the expression of claudins in neoplasm as well as in adjacent normal mucosa in each slide. RESULTS:Immunohistochemical staining with claudins antibodies had membranous pattern; claudins expression in adjacent normal mucosa was uniformly close to maximum. Serrated lesions showed the lowest level of expression of claudin-1 among other groups (p<0,05). In the group of adenocarcinomas we found moderate negative correlation between claudin-1 expression level and grade of adenocarcinoma. Claudin-3 expression level was significantly higher in adenocarcinomas compared to serrated lesions (p=0,025) and in conventional adenomas compared to serrated lesions (p=0,034). Expression of claudin-4 was strong in most cases, except for tubular adenomas that showed moderate expression in most cases. CONCLUSION:We found no statistically significant difference between levels of expression of claudin-1, claudin-3 and claudin-4 expression levels among adenocarcinomas, hyperplastic polyps, sessile serrated adenomas, traditional serrated adenomas, tubular and tubular-villous adenomas. But we detected significant difference after enlargement of the groups. This fact may argue for general development pathway of hyperplastic polyps and sessile serrated adenomas, and of tubular and tubular-villous adenomas. Expression of claudin-1 and claudin-3 revealed difference of serrated lesions from conventional adenomas and adenocarcinomas, that confirms conception of independent «serrated» pathway of cancerogenesis.
FH535 inhibits proliferation and migration of colorectal cancer cells by regulating CyclinA2 and Claudin1 gene expression.
Tu Xuezi,Hong Dan,Jiang Yiyan,Lou Zhefeng,Wang Keke,Jiang Yiwen,Jin Longjin
Wnt signaling pathway plays a major role in the progression of colorectal cancer (CRC). Small molecules which can cut off this signal transduction can be promising anti-cancer drugs for CRC therapy. Therefore, we aimed to investigate the mechanisms of FH535, an inhibitor of the Wnt signaling pathway, on inhibiting proliferation and migration of colorectal cancer cells DLD-1 and SW620. We found that FH535 could significantly suppress the growth of DLD-1 and SW620 cells in a concentration-dependent and time-dependent manner. The results of cell cycle tests showed that FH535 could significantly induce G2/M arrest in colorectal cancer cells. Transwell and Wound-healing assays revealed that FH535 notably inhibited cell migration. Moreover, we found that FH535 down-regulated β-catenin and CyclinA2 expressions while up-regulating Claudin-1 expression at both mRNA and protein levels, which may contribute to the FH535-induced inhibitory effect on proliferation and migration in human colorectal cancer cells. Our study revealed that FH535 inhibited proliferation and migration of colorectal cancer cells by regulating CyclinA2 and Claudin1 gene expression, which enriches regulatory network of FH535 and may contribute to being promising anti-cancer drugs for CRC therapy.
Claudin-7 downregulation induces metastasis and invasion in colorectal cancer via the promotion of epithelial-mesenchymal transition.
Wang Kun,Li Tengyan,Xu Chang,Ding Yuhan,Li Wenjing,Ding Lei
Biochemical and biophysical research communications
The dysregulation of the tight junctions (TJs) protein claudin-7 is closely related to the development and metastasis of colorectal cancer (CRC). The aim of this study was to investigate the expression of claudin-7 and characterize the relationship between claudin-7 expression and epithelial-mesenchymal transition (EMT) in CRC. In this study, the expression of claudin-7, E-cadherin, vimentin and snail-1 was detected by immunohistochemistry (IHC) in a set of 80 CRC specimens comprising 20 specimens each of well-differentiated, moderately differentiated, poorly differentiated and liver metastases tissues. The correlation between claudin-7 and EMT-related proteins in the stably transfected claudin-7 knockdown HCT116 cell line was analyzed by IHC, immunofluorescence (IF), Western blotting (WB) and nude mouse xenograft models. The results revealed that the expression of claudin-7 was downregulated as CRC tissue differentiation grade decreased, and that low claudin-7 expression corresponded to the downregulation of E-cadherin (r = 0.725, p < 0.001) and upregulation of vimentin (r = -0.376, p = 0.001) and snail-1 (r = -0.599, p < 0.001). Additionally, in the claudin-7 knockdown HCT116 cell line, the staining intensity and expression of E-cadherin was decreased, while the immunoreactivity and expression of vimentin and snail-1 was increased. Futhermore, the result of tumor formation experiment was consistent with CRC tissues. In conclusion, the expression of claudin-7 in CRC is downregulated as differentiation grade decreases. Claudin-7 downregulation may promote the invasion and metastasis of CRC by regulating EMT. Our results provide new perspectives for a potential therapeutic target for CRC.
Treatment-related survival associations of claudin-2 expression in fibroblasts of colorectal cancer.
Mezheyeuski Artur,Strell Carina,Hrynchyk Ina,Guren Tormod Kyrre,Dragomir Anca,Doroshenko Tatyana,Pashkova Oksana,Gorgun Julia,Ruksha Kseniya,Pfeiffer Per,Kure Elin H,Sorbye Halfdan,Edler David,Martling Anna,Glimelius Bengt,Östman Arne,Portyanko Anna
Virchows Archiv : an international journal of pathology
Claudin-2 is a trans-membrane protein-component of tight junctions in epithelial cells. Elevated claudin-2 expression has been reported in colorectal cancer (CRC). The aim of this study was to investigate the expression patterns of claudin-2 in human CRC samples and analyze its association with clinical characteristics and treatment outcome. TMAs of primary tumors from two cohorts of metastatic CRC (mCRC) were used. Claudin-2 IHC staining was evaluated in a semi-quantitative manner in different regions and cell types. Claudin-2 expression was also analyzed by immunofluorescence in primary cultures of human CRC cancer-associated fibroblasts (CAFs). Initial analyses identified previously unrecognized expression patterns of claudin-2 in CAFs of human CRC. Claudin-2 expression in CAFs of the invasive margin was associated with shorter progression-free survival. Subgroup analyses demonstrated that the survival associations occurred among cases that received 5-FU+oxaliplatin combination treatment, but not in patients receiving 5-FU±irinotecan. The finding was validated by analyses of the independent cohort. In summary, previously unreported stromal expression of claudin-2 in CAFs of human CRC was detected together with significant association between high claudin-2 expression in CAFs and shorter survival in 5-FU+oxaliplatin-treated mCRC patients.
Expression of claudin 1, claudin 4, and claudin 7 in colorectal cancer and its relation with CLDN DNA methylation patterns.
Hahn-Strömberg Victoria,Askari Shlear,Ahmad Abrar,Befekadu Rahel,Nilsson Torbjörn K
Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine
Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p ≤ 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.